Parathyroid Hormone Receptor 1 (PTH1R) Antibodies and Uses Thereof

ABSTRACT

The present disclosure relates, in general, human antibodies against human parathyroid hormone receptor 1 (PTH1R) and methods of use of such antibodies in the treatment of cancer, Humoral Hypercalcemia of Malignancy (HHM), or Primary Hyperparathyroidism (PHPT) and Secondary Hyperparathyroidism (SHPT) and cachexia.

This application claims the priority benefit of U.S. Provisional PatentApplication No. 62/369,745, filed Aug. 1, 2016, U.S. Provisional PatentApplication No. 62/432,338, filed Dec. 9, 2016 and U.S. ProvisionalPatent Application No. 62/479,637, filed Mar. 31, 2017, hereinincorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: Filename: 50986_SeqListing.txt; Size: 108,540bytes, created on Jul. 28, 2017.

FIELD OF THE INVENTION

The present disclosure relates, in general, to parathyroid hormonereceptor 1 (PTH1R) antibodies and therapy for treating conditionsassociated with increased parathyroid hormone expression, increasedparathyroid hormone related protein expression or PTH1R expressioncomprising administering to a subject in need thereof a therapeuticallyeffective amount of an PTH1R antibody.

BACKGROUND

G protein-coupled receptors (GPCRs) are a group of plasma membranereceptors that transduce signals from extracellular ligands to signalsin intracellular relay proteins, the heterotrimeric GTP binding proteins(G proteins). GPCRs represent one of largest and most diverse proteinfamilies in mammalian genomes. (Hutchings et al., 2010 mAbs 2:6,594-606; Strader et al., 1994 Annual Review of Biochemistry 63:101-132).GPCRs are characterized by having an extracellular N-terminus,7-transmembrane spanning (TM) domains and an intracellular C-terminus(Hutchings et al., 2010 mAbs 2:6, 594-606). In vertebrates, thesereceptors can be classified into families based on their sequencesimilarity within the 7 TM domains, Class 1 (rhodopsin-like family),Class 2 (secretin and adhesion family), Class 3 (the glutamate family),and Class F (frizzled family) (Hutchings et al., 2010 mAbs 2:6, 594-606;Fredriksson et al., 2003 Mol Pharmacol. 63(6):1256-72).

The parathyroid hormone receptor (PTHR) is a class 2 GPCR that activatesthe adenylyl cyclase/cAMP signaling pathway. Similar to all class 2GPCRs that bind peptide hormones, the PTHR has a relatively large (˜160amino acid)N-terminal extracellular domain, herein termed the N domain,that plays a major role in hormone binding (Shimizu et al., 2004 J BiolChem. 280(3):1797-807). Two types of PTHR, type 1 (PTH1R) and type 2(PTH2R) have been identified. PTH1R mediates the actions of twopolypeptide ligands; parathyroid hormone (PTH), an endocrine hormonethat regulates the levels of calcium and inorganic phosphate in theblood by acting on bone and kidney, and PTH-related protein (PTHrP), aparacrine-factor that regulates cell differentiation and proliferationprograms in developing bone and other tissues (Gardella et al., 2015Pharmacol Rev. 67(2): 310-337). PTH a principal regulator of boneremodeling and calcium ion homeostasis, exerts its effects by bindingand activating the PTHR (Shimizu et al., 2004 J Biol Chem.280(3):1797-807).

SUMMARY OF THE INVENTION

The invention provides materials, methods, and uses relating toantibodies against parathyroid hormone receptor 1 (PTH1R). Thedisclosure provides antibodies that bind PTH1R. In particular, thepresent disclosure provides methods of use of such antibodies in thetreatment of cancer, Humoral Hypercalcemia of Malignancy (HHM), PrimaryHyperparathyroidism (PHPT), Secondary Hyperparathyroidism (SHPT) andcachexia.

In various embodiments, the disclosure provides an antibody specific forPTH1R with an affinity K_(d) of 10⁻⁶ M or less. In various embodiments,the disclosure provides an antibody specific for PTH1R with an affinityK_(d) of 2×10⁻⁶ M or less. In exemplary embodiments, an anti-PTH1Rantibody described herein binds at least with an affinity of 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or less. In other embodiments, an antibodydescribed herein binds to PTH1R with at least 2-50 fold, 10-100 fold,2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%,50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higheraffinity (e.g., preferentially binds to PTH1R) compared to binding toPTH2R. In certain embodiments, the affinity is measured by surfaceplasmon resonance or KinExA assay.

In a related aspect, the antibody binds the N-terminal portion of PTH1R.In a further aspect, the disclosure contemplates an antibody that doesnot bind parathyroid hormone receptor 2 (PTH2R). In various aspects theantibody binds PTH1R on the surface of a cell. In certain aspects theantibody binds allosterically to PTH1R.

In a preferred embodiment, the antibody is a negative modulatorantibody, optionally wherein the antibody is capable of weakening thebinding affinity between PTH or PTHrP and with PTH1R by at least about2-fold, optionally up to 1000-fold. In other embodiments, an antibodydescribed herein is capable of weakening the binding affinity betweenPTH or PTHrP with at least 2-1000 fold, 10-100 fold, 2-fold, 5-fold,10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold,500-fold, 600-fold, 700-fold, 800-fold, 900-fold or 1000-fold.

In a various embodiments, the antibody inhibits calcium flux in a cellin response to stimulation of the receptor with parathyroid hormone(PTH) or parathyroid hormone related protein (PTHrP). In relatedembodiments, the antibody inhibits PTH- or PTHrP-mediated cyclicadenosine mono-phosphate (cAMP) accumulation.

In one embodiment, the PTH1R antibody is a monoclonal antibody.

In one aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising (a) a heavy chaincomplementary determining repeat (CDR)1 amino acid sequence set forth inFIG. 21 or SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60,63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variantthereof in which one or two amino acids have been changed; (b) a heavychain CDR2 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 28,31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82,85, 88, 91, 94, 97, 100, 103 that is from the same heavy chain variableregion as (a), or a variant thereof in which one or two amino acids havebeen changed; and (c) a heavy chain CDR3 amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59,62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that isfrom the same heavy chain variable region as (a), or a variant thereofin which one or two amino acids have been changed.

In a related aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising: (a) a heavy chainCDR1 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 27, 30, 33,36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87,90, 93, 96, 99, 102, or a variant thereof having at least 70% identitythereto; (b) a heavy chain CDR2 amino acid sequence set forth in SEQ IDNOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76,79, 82, 85, 88, 91, 94, 97, 100, 103 that is from the same heavy chainvariable region as (a), or a variant thereof having at least 70%identity thereto; and (c) a heavy chain CDR3 amino acid sequence setforth in SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65,68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that is from thesame heavy chain variable region as (a), or a variant thereof having atleast 70% identity thereto.

In a further aspect, the disclosure contemplates an antibody that bindsan antibody that binds parathyroid hormone receptor 1 (PTH1R)comprising: (a) a heavy chain CDR1 amino acid sequence set forth in SEQID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72,75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variant thereof having atleast 70% identity thereto; (b) an independently selected heavy chainCDR2 amino acid sequence set forth in in FIG. 21 or SEQ ID NOs: 28, 31,34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85,88, 91, 94, 97, 100, 103, or a variant thereof having at least 70%identity thereto; and (c) an independently selected heavy chain CDR3amino acid sequence set forth in in FIG. 21 or SEQ ID NOs: 29, 32, 35,38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89,92, 95, 98, 101, 104, or a variant thereof having at least 70% identitythereto.

In certain embodiments, at least two of the heavy chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:27-104. In a related embodiment, three of the heavy chain CDR1, CDR2 andCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:27-104.

In some embodiments, an antibody contemplated herein comprises an aminoacid sequence at least 85% identical to a heavy chain variable regionamino acid sequence set forth in FIG. 21 or SEQ ID NOs: 1-26. In someembodiments, provided herein is an antibody that comprises an amino acidsequence at least 95% identical to a heavy chain variable region aminoacid sequence set forth in FIG. 21 or SEQ ID NOs: 1-26.

It is further contemplated that an antibody described herein comprises apolypeptide sequence having an amino acid sequence at least 70%identical over all three HCDRs in a heavy chain variable region, theamino acid sequences of HCDR1, HCDR2 and HCDR3 set forth in FIG. 21 orSEQ ID NOs: 27-104.

In certain embodiments, an antibody contemplated herein comprises one ormore heavy chain framework amino acids have been replaced withcorresponding amino acid(s) from another human antibody amino acidsequence.

In one embodiment, an antibody contemplated herein further comprises anyone of the light chain CDR amino acid sequences set forth in FIG. 21 orSEQ ID NOs: 131-208. In some embodiments, an antibody comprises at leasttwo of the light chain CDR amino acid sequences set forth in FIG. 21 orSEQ ID NOs: 131-208. In other embodiments, an antibody comprises atleast three of the light chain CDR amino acid sequences set forth inFIG. 21 or SEQ ID NOs: 131-208.

In another aspect, an antibody described herein comprises (a) a lightchain CDR1 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 131,134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173,176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or a variantthereof in which one or two amino acids have been changed, or aconsensus sequence thereof set out in FIG. 21; (b) a light chain CDR2amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 132, 135, 138,141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180,183, 186, 189, 192, 195, 198, 201, 204, 207 that is from the same lightchain variable region as (a), or a variant thereof in which one or twoamino acids have been changed, or a consensus sequence thereof set outin FIG. 21; and (c) a light chain CDR3 amino acid sequence set forth inFIG. 21 or SEQ ID NOs: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160,163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202,205, 208 that is from the same light chain variable region as (a), or avariant thereof in which one or two amino acids have been changed, or aconsensus sequence thereof set out in FIG. 21.

In alternative embodiments, an antibody contemplated herein comprises:(a) a light chain CDR1 amino acid sequence set forth in FIG. 21 or SEQID NOs: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167,170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or avariant thereof in which one or two amino acids have been changed; (b)an independently selected light chain CDR2 amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 132, 135, 138, 141, 144, 147, 150, 153, 156,159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198,201, 204, 207, or a variant thereof in which one or two amino acids havebeen changed; and (c) an independently selected light chain CDR3 aminoacid sequence set forth in FIG. 21 or SEQ ID NOs: 133, 136, 139, 142,145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184,187, 190, 193, 196, 199, 202, 205, 208, or a variant thereof in whichone or two amino acids have been changed.

In certain embodiments, at least two of the light chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:131-208.

It is further contemplated that an antibody described herein comprisesan amino acid sequence at least 70% identical to a light chain variableregion amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 105-130.In a related embodiment, the antibody comprises an amino acid sequenceat least 85% identical to a light chain variable region amino acidsequence set forth in FIG. 21 or SEQ ID NOs: 105-130. In a furtherembodiment, the antibody comprises an amino acid sequence at least 95%identical to a light chain variable region amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 105-130. In still another embodiment, theantibody comprises a light chain variable region amino acid sequence setforth in FIG. 21 or SEQ ID NOs: 105-130.

In a further embodiment, an antibody described herein comprises apolypeptide sequence having an amino acid sequence at least 70%identical over all three LCDRs of a light chain variable region, theamino acid sequences of LCDR1, LCDR2 and LCDR3 set forth in FIG. 21 orSEQ ID NOs: 131-208.

In certain embodiments, an antibody described herein comprises (i) anamino acid sequence at least 70% identical over all three LCDRs, of alight chain variable region, the amino acid sequences of LCDR1, LCDR2and LCDR3 set forth in FIG. 21 or SEQ ID NOs: 131-208 and (ii) an aminoacid sequence at least 70% identical over all three HCDRs of a heavychain variable region, the amino acid sequences of HCDR1, HCDR2 andHCDR3 set forth in FIG. 21 or SEQ ID NOs: 27-104.

In another aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising a light chain variableregion and/or a heavy chain variable region, wherein (a) the light chainvariable region comprises at least a CDR1 selected from SEQ ID NOs: 131,134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173,176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 or sequences atleast 80% identical thereto, a CDR2 selected from SEQ ID NOs: 132, 135,138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177,180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or sequences at least80% identical thereto, and/or a CDR3 selected from SEQ ID NOs: 133, 136,139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178,181, 184, 187, 190, 193, 196, 199, 202, 205, 208 or sequences at least80% identical thereto; and/or wherein (b) the heavy chain variableregion comprises at least a CDR1 selected from SEQ ID NOs: 27, 30, 33,36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87,90, 93, 96, 99, 102 or sequences at least 80% identical thereto, a CDR2selected from SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58,61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71,74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 80%identical thereto.

In a related embodiment, an antibody described herein comprises (a) alight chain variable region comprising at least a CDR1 selected from SEQID NO: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167,170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168,171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 orsequences at least 90% identical thereto, and a CDR3 selected from SEQID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169,172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 orsequences at least 90% identical thereto; and/or wherein (b) the heavychain variable region comprises at least a CDR1 selected from SEQ ID NO:27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78,81, 84, 87, 90, 93, 96, 99, 102 or sequences at least 90% identicalthereto, a CDR2 selected from SEQ ID NO: 28, 31, 34, 37, 40, 43, 46, 49,52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103or sequences at least 90% identical thereto, and a CDR3 selected fromSEQ ID NO: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71,74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 90%identical thereto.

In some embodiments, an antibody of the disclosure further comprises aheavy chain constant region, wherein the heavy chain constant region isa modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, orcombinations thereof.

In certain embodiments, an antibody is provided in which one or morelight chain framework amino acids have been replaced with correspondingamino acid(s) from another human antibody amino acid sequence,optionally wherein the framework comprises one or more of the changesset out in FIGS. 1A and 1B.

In one aspect, the antibody of the disclosure is selected from the groupconsisting of XPA.85.012, XPA.85.017, XPA.85.288, XPA.85.328,XPA.85.329, XPA.85.330 and XPA.85.349.

In one embodiment, an antibody described herein further comprises ahuman light chain constant region attached to said light chain variableregion. In some embodiments, the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof.

In another aspect, the disclosure provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the heavy chainor light chain as described herein. In various embodiments thenucleotide sequences encoding the antibody variable regions are set outin SEQ ID NOs: 209-234 (heavy chain) and 235-260 (light chain).

In various embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the light chain variable region amino acidsequence of any one of SEQ ID NOs: 105-130 or a fragment thereof. In oneembodiment, the nucleic acid molecule comprises the light chain variableregion nucleotide sequence of any one of SEQ ID NOs: 235-260, or afragment thereof. In further embodiments, the nucleic acid moleculecomprises a nucleotide sequence that encodes the heavy chain variableregion amino acid sequence of any one of SEQ ID NOs: 1-26 or a fragmentthereof. In one embodiment, the nucleic acid molecule comprises theheavy chain variable region nucleotide sequence of any one of SEQ IDNOs: 209-234, or a fragment thereof. Nucleic acid molecules of thedisclosure further include all nucleic acid sequences, including thesequences in SEQ ID NOs: 209-260, and nucleic acid sequences comprisingdegenerate codons based on the diversity of the genetic code, encodingan amino acid sequence of the heavy and light chain variable regions ofan antibody described herein or any HCDRs or LCDRs described herein, andencoding the CDR amino acid sequences as set out in SEQ ID NOs: 27-104and 131-208, as well as nucleic acids that hybridize under highlystringent conditions, such as those described herein, to a nucleic acidsequence encoding an amino acid sequence of the heavy and light chainvariable regions of an antibody described herein set out in SEQ ID NOs:1-26 or 105-130 or any HCDRs or LCDRs described herein as set out in SEQID NOs: 27-104 or 131-208.

In some embodiments, the nucleic acid molecule encodes a VL amino acidsequence that is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96 97, 98 or 99% identical to a VL amino acid sequence set out inSEQ ID NOs: 105-130. In a related aspect, the VL amino acid sequence isa consensus sequence. Nucleic acid molecules of the disclosure includenucleic acids that hybridize under highly stringent conditions, such asthose described herein, to a nucleic acid sequence encoding the lightchain variable region amino acid sequence of SEQ ID NOs: 105-130, orthat has the light chain variable region nucleic acid sequence of SEQ IDNOs: 235-260. In some embodiments, the nucleic acid encodes the aminoacid sequence of the heavy chain CDRs of said antibody set out in SEQ IDNO: 131-208.

It is further contemplated that a nucleic acid molecule of thedisclosure comprises a nucleotide sequence that encodes the VH aminoacid sequence of any one of antibodies described herein, or a fragmentthereof. In some embodiments, the nucleic acid encodes the amino acidsequence of the heavy chain and/or light chain CDRs of said antibody. Insome embodiments, said fragment is a contiguous fragment comprisingheavy chain and/or light chain CDR1-CDR3. In one embodiment, saidfragment comprises at least one, two or three of a heavy chain and/orlight chain CDR1, CDR2, or CDR3 region, optionally with a differenthuman or human consensus framework, and optionally with 1, or up to 2,or up to 3 mutations in the CDRs. CDR amino acid sequences are set outin SEQ ID NOs: 27-104 and 131-208.

In a related aspect, the nucleic acid molecule comprises a nucleotidesequence that encodes the heavy chain variable region amino acidsequence of one of heavy chain of SEQ ID NOs: 1-26, or a fragmentthereof. In one embodiment, the nucleic acid molecule comprises theheavy chain variable region having a nucleotide sequence set out in SEQID NOs: 209-234, or a fragment thereof.

In some embodiments, the nucleic acid molecule encodes a VH amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a VH amino acid sequence set out in SEQ ID NOs: 1-26.In a related aspect, the VH amino acid sequence is a consensus sequence.Nucleic acid molecules of the disclosure further include nucleic acidsthat hybridize under highly stringent conditions, such as thosedescribed herein, to a nucleic acid sequence encoding the heavy chainvariable region amino acid sequence of SEQ ID NOs: 1-26, or that has theheavy chain variable region nucleic acid sequence of any one of SEQ IDNOs: 209-234.

It is further contemplated that the nucleic acids of the disclosure mayencode a full-length light chain or heavy chain of an antibody selectedfrom the antibodies set out in FIG. 21 wherein a full-length light chainor full-length heavy chain comprises a light chain constant region or aheavy chain constant region, respectively, light chain constant regionsoptionally include unmodified or modified kappa or lambda regions, andheavy constant regions include unmodified or modified constant regionsof any of the classes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, orIgE. Modified IgG4 constant regions include constant regions containingone or more of the mutations Ser228Pro and Leu235Glu (Angal et al.,(1993) Mol. Immunol. 30: 105-108; Reddy et al., (2000) J. Immunol. 164:1925-1933).

In one aspect, the full length variable light chain antibody comprisesthe amino acid sequences set out in SEQ ID NOs: 105-130. It is furthercontemplated that the nucleotide sequence encoding the full-length lightchain encodes the amino acid sequences set out in SEQ ID NOs: 105-130and comprises the nucleotide sequences set forth in SEQ ID NOs: 235-260.

In one aspect, the full length variable heavy chain antibody comprisesthe sequences in any one of SEQ ID NOs: 1-26. Further provided arenucleotide sequences that encode the full-length heavy chain variableregion amino acid sequences set out in SEQ ID NOs: 1-26, and comprisethe nucleotide sequences set forth in any one of SEQ ID NOs: 209-234.

In a further aspect, the disclosure provides an expression vectorcomprising a nucleic acid molecule contemplated herein operably linkedto an expression control sequence. Also contemplated is a host cellcomprising an expression vector or a nucleic acid molecule of thedisclosure. In certain embodiments, the disclosure provides a host cellcomprising a nucleic acid molecule encoding a heavy chain and a lightchain variable region, wherein the heavy chain and light chain nucleicacids are expressed by different nucleic acids or on the same nucleicacid.

In a related aspect, the disclosure provides a method of using the hostcell as described herein to produce an antibody, the method comprisingculturing the host cell under suitable conditions and recovering saidantibody. Also provided is an antibody produced by the method disclosedherein.

The disclosure further contemplates a sterile pharmaceutical compositioncomprising the antibody as disclosed herein and a pharmaceuticallyacceptable carrier.

In another aspect, the disclosure provides a method for treatinghypercalcemia associated with increased parathyroid hormone orparathyroid hormone related protein expression comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or a pharmaceutical composition contemplatedherein.

In another aspect, the disclosure provides a method for treating adisease, condition or disorder associated with increased parathyroidhormone expression, increased parathyroid hormone related proteinexpression or increased PTH1R expression comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or a pharmaceutical composition contemplatedherein.

In another aspect, the disclosure provides a method for treating adisease, condition or disorder is selected from the group consisting ofcancer, PTH- or PTHrP-induced hypercalcemia, Humoral Hypercalcemia ofMalignancy (HHM), familial hypocalciuric hypercalcemia, tuberculosis,sarcoidosis, Primary Hyperparathyroidism (PHPT), SecondaryHyperparathyroidism (SHPT) and cachexia.

In various embodiments, the disease is PHPT and the subject is anon-surgical patient or a patient who has failed surgery. In variousembodiments, the disease is SHPT and the subject has chronic kidneydisease.

In various embodiments, the disclosure provides a method for treatingcancer wherein the administration reduces the incidence of cancermetastasis in the subject compared to a subject not receiving theantibody.

In a related aspect, the disclosure provides a method for treatingcancer comprising administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein. In certain embodiments, the cancer isselected from the group consisting of bone cancer, lung cancer,hepatocellular cancer, pancreatic cancer, kidney cancer, fibroticcancer, breast cancer, myeloma squamous cell carcinoma, colorectalcancer and prostate cancer. In related aspects the cancer is metastatic.In a related aspect, the metastasis includes metastasis to the bone orskeletal tissues, liver, lung, kidney or pancreas.

In various embodiments, administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein ameliorates one or more symptoms ofhypercalcemia.

In various embodiments, administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein ameliorates one or more symptoms ofwasting syndrome or cachexia in PTHrP induced HHM.

In various embodiments, administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein extends HHM survival due to reducedhypercalcemia and/or wasting syndrome.

In various embodiments, the antibody disclosed herein is administeredintravenously, intraarterially, intraperitoneally, intramuscularly,intradermally or subcutaneously. In related embodiments, the antibodydisclosed herein is administered in combination with a second agent. Inrelated embodiments, the antibody disclosed herein is administered onceper week, once every 2 weeks, twice per month, once monthly, once everytwo months, or once every three months.

In various embodiments, the antibody compositions can be administeredintravenously, subcutaneously or intramuscularly, in a dose range of0.3-30 mg/kg twice weekly or every 1, 2 or 4 weeks. In variousembodiments, the dose can be 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 30mg/kg. In various embodiments, the antibody compositions can beadministered intravenously in a dose range of 0.3-3 mg/kg, 1 to 6 mg/kg,or 2 to 6 mg/kg twice weekly or every 1, 2 or 4 weeks. Alternatively,the antibody compositions can be administered intravenously,subcutaneously or intramuscularly in a dose range of 0.5-5 mg/kg twiceweekly or every 1, 2 or 4 weeks.

In various embodiments, the disclosure provides a method foradministering a composition comprising an antibody or a pharmaceuticalcomposition disclosed herein in the treatment a condition or disorderassociated with increased parathyroid hormone expression or increasedparathyroid hormone related protein expression.

In various embodiments, the disclosure provides a method foradministering a composition comprising an antibody or a pharmaceuticalcomposition disclosed herein in the treatment a condition or disorderassociated with hypercalcemia.

In one aspect, the PTH1R antibody is a monoclonal antibody (or activefragments thereof) that can cross react, detect, bind to and neutralizePTH1R from multiple species (e.g. the human PTH1R antibody disclosedherein can cross react, detect, bind to and neutralize mouse PTH1R.Mouse PTH1R antibody disclosed herein can cross react, detect, bind toand neutralize human PTH1R).

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the invention and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment”, “some embodiments”, “certain embodiments”, “furtherembodiment”, “specific exemplary embodiments”, and/or “anotherembodiment”, each of these types of embodiments is a non-limitingexample of a feature that is intended to be combined with any otherfeature, or combination of features, described herein without having tolist every possible combination. Such features or combinations offeatures apply to any of the aspects of the invention. Where examples ofvalues falling within ranges are disclosed, any of these examples arecontemplated as possible endpoints of a range, any and all numericvalues between such endpoints are contemplated, and any and allcombinations of upper and lower endpoints are envisioned.

The headings herein are for the convenience of the reader and notintended to be limiting. Additional aspects, embodiments, and variationsof the invention will be apparent from the Detailed Description and/orDrawing and/or claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: A multiple sequence alignment of the light chains ofselected clones against the parent sequence. Multiple sequencealignments (MSA) of the light chain amino acids of clones relating toXPA.85.012 (FIG. 1A) or from clones relating to XPA.85.017 (FIG. 1B),were performed against the parent sequence using the ClustalW algorithm.This alignment was used to generate a phylogenetic tree using CLCBioSequence viewer software. The tree was exported in Newick format. TheMSA was exported in FASTA format and run through the EMBOSS programShowalign to convert the alignment to dot-identity format. A script waswritten using the R language to combine the tree with the correspondingdot-identify alignment sequence, color-coded by light chain family. Thebranch line formats also correspond to light chain family to make iteasy to read in B/W format. In the displayed alignment, the parentsequence is shown completely. Where other sequences are identical, theresidue is shown as a dot. The differences are displayed. CDR regionsidentified using the IMGT numbering system are called out by boxes onthe alignment. Framework differences from the parent are identified.This was done by running the parental light chain through a softwareprogram (PHEnom; XOMA (US) LLC)) and looking at the position frequencymatrix that was displayed in the results. If a framework change in asequence was toward a canonical amino acid, it is shown as (+), if awayfrom canonical sequence (in terms of frequency) it is shown as (−). Ifthe residue is not seen in the frequency matrix, it is an amino acidrarely used in that position and it is indicated by an (R) and the aminoacid is indicated with square brackets. When the sequences belong to adifferent family that the parental, the family member closest to theparental is used as the “seed” in this PHEnom analysis.

FIGS. 2A-2D: Representative SPR derived kinetic data comparing theparental clones XPA.85.012 (FIG. 2B) and XPA.85.017 (FIG. 2D) with theirrespective higher affinity light chain variants XPA.85.288 (FIG. 2A) andXPA.85.287 (FIG. 2C) respectively. The IgGs were captured to the sensorsurface via the Fc region and recombinant PTH1R N-terminal ECD wasinjected at 200, 50, 12.5, and 3.125 nM as described in the examplemethod.

FIGS. 3A-3B: Binding Curves of XPA.85.012 and XPA.85.017 on CHOK1 (opencircles) and CHO human PTH1R (solid squares) cells. Binding Curves ofXPA.85.012 (FIG. 3A). Binding Curves of XPA.85.017 (FIG. 3B). Bothantibodies bind specifically to PTH1R expressed on CHO human PTH1Rcells.

FIGS. 4A-4B: XPA.85.012 and XPA.85.017 Binding in the Presence of PTH orPTHrP. Increasing concentrations of anti-PTH1R antibodies were incubatedwith CHO human PTH1R cells in the presence or absence of saturatingconcentrations of PTH or PTHrP. Antibody binding was detected withR-Phycoerythrin anti-human IgG antibody alone (open circles), antibodywith 2.5 μM PTH (solid squares), and antibody with 2.5 μM PTHrP (soliddiamond). Binding Curves of XPA.85.012 (FIG. 4A). Binding Curves ofXPA.85.017 (FIG. 4B). Both antibodies bound to PTH1R in the presence ofeither PTH or PTHrP, though antibody binding to PTH1R was reduced in thepresence of either ligand.

FIGS. 5A-5B: On-Rate of XPA.85.012 and XPA.85.017 by Flow Cytometry.Increasing antibody concentrations were incubated with CHO human PTH1Rcells for a certain amount of time. Antibody binding was detected withanti-human IgG APC. A time course was run for XPA.85.017 due to its slowon rate when compared to XPA.85.012 (FIG. 5A). In the graph, as theincubation time increases, the antibody binding curve is shifted to theleft. This shows that the antibody has not reached equilibrium and ittakes time for the antibody to bind. XPA.85.012 has a faster on-rate(FIG. 5B). Antibody binding at 1 hr (open circles) has a similar EC50value as the 24 hr incubation time point (solid hexagon). XPA.85.012 canreach binding equilibrium within 1 hr whereas it can take XPA.85.017 24hr to reach binding equilibrium. Once both antibodies reach equilibrium,they have similar EC50 values.

FIGS. 6A-6D: XPA.85.012 and XPA.85.017 species cross-reactivity anddifferential binding profiles. Increasing concentrations of anti-PTH1Rantibodies were incubated for 24 hr with cells expressing PTH1Rorthologs in the presence or absence of saturating concentration of PTHor PTHrP. Antibody binding was detected with R-Phycoerythrin anti-humanIgG. Both XPA.85.012 and XPA.85.017 were cross-reactive to the differentPTH1R orthologs and both antibodies bound better in the absence of theligands, PTH (square symbol) or PTHrP (diamond symbol). Antibodiesbinding to CHO cells expressing human PTH1R+/−PTH (FIG. 6A) or +/−PTHrP(FIG. 6B). Antibodies binding to CHO cells expressing mouse PTH1R+/−PTH(FIG. 6C) or +/−PTHrP (FIG. 6D).

FIGS. 7A-7C: Antibodies binding to Saos-2 overexpressing human PTH1R inthe Presence or Absence of PTH and PTHrP. Increasing concentrations ofanti-PTH1R antibodies were incubated with Saos-2 human PTH1R cells inthe presence or absence of saturating concentrations of PTH or PTHrP.XPA.85.328 (FIG. 7A), XPA.85.329 (FIG. 7B), XPA.85.330 (FIG. 7C).Antibody binding was detected with R-Phycoerythrin anti-human IgG.Antibody alone (open circles) antibody with 1 μM PTH (solid squares),and antibody with 3 μM PTHrP (solid diamond). All three antibodiesshowed reduced binding to PTH1R in the presence of either PTH or PTHrP.

FIG. 8: Anti-PTH1R antibodies bind specifically to PTH1R. Increasingconcentrations of anti-PTH1R antibodies were added to CHO cellsoverexpressing human PTH2R and incubated for 1 hr at 4° C. (FIG. 8).Cells were washed twice with FACS Buffer and antibody binding wasdetected with anti-human IgG APC. None of the antibodies tested showedspecific binding to CHO cells overexpressing human PTH2R. Anti-KLH.G2was used a negative control for binding. All the tested antibodies werespecific to PTH1R.

FIGS. 9A-9D: Anti-PTH1R antibodies inhibit ligand-mediated cAMPaccumulation and calcium mobilization. Human PTH1R CHOK1 cells werepreincubated for 30 min at 37° C. with 267 nM (40 μg/ml) of eitherXPA.85.012 (solid squares) or XPA.85.017 (solid triangles), followed byinduction with increasing concentrations of either PTH(1-34) (FIGS. 9Aand 9C) or PTHrP(1-34) (FIGS. 9B and 9D). Both antibodies inhibitedligand-mediated cAMP accumulation (Gs/PKA pathway, FIGS. 9A and 9B) andcalcium mobilization (Gq/PKC pathway, FIGS. 9C and 9D).

FIGS. 10A-10D: Following affinity engineering of XPA.85.017 (opencircles) by light chain shuffling, CHOK1 cells stably overexpressingeither human (FIGS. 10A and 10B). or mouse PTH1R (FIGS. 10C and 10D)were preincubated for 30 min at 37° C. with 267 nM (40 μg/ml) of variantIgGs, followed by induction with increasing concentrations of PTH(1-34)for 45 min at 37° C. Variant IgGs exhibited a range of inhibition ofligand-mediated cAMP accumulation (Gs/PKA pathway) via human and mousePTH1R.

FIGS. 11A-11D Comparison of variant and parental IgGs activities in cAMPassays using SaOS-2 and UMR106 cells, which endogenously express nativehuman and rat PTH1R, respectively. IgGs (267 nM or 40 μg/ml) werepreincubated with cells for 30 min at 37° C. prior to induction of cAMPaccumulation by PTH(1-34) or PTHrP(1-34) for 45 min at 37° C. Data arepresented as mean+SEM, and represent the average of RLUs measured insinglicate (FIGS. 11A and 11D) or duplicate (FIGS. 11B and 11C) wells.Affinity engineering of XPA.85.017 by light chain shuffling resulted inXPA.85.287. This variant showed a significant improvement in activityagainst human PTH1R (FIG. 11B) (open squares) versus parent (solidsquares), with little change in activity against rat PTH1R (FIG. 11C).In contrast, affinity engineering of XPA.85.012 (solid triangles)resulted in a variant, XPA.85.288 (open triangles), showing asignificant improvement in activity against both native human PTH1R(FIG. 11A), as well as native rat PTH1R (FIG. 11C). The XPA.85.288 wasalso found to significantly inhibit PTHrP-induced cAMP in SaOS-2 cellsexpressing native human PTH1R (FIG. 11D).

FIGS. 12A-12B: Concentration-response curves for PTHrP(1-34) andincreasing concentrations of XPA.85.287 (an affinity engineered variantof XPA.85.017) in cAMP assays using Human PTH1R CHOK1 (FIG. 12A) andMouse PTH1R CHOK1 (FIG. 12B) cells. Schild regression analyses wereperformed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego,Calif.), revealing non-parallel rightward shifts inconcentration-response curves. These results are consistent with anon-competitive (allosteric) mechanism of action of the variant IgG.

FIGS. 13A-13B: Concentration-response curves for PTHrP(1-34) andincreasing concentrations of XPA.85.288 (an affinity engineered variantof XPA.85.012) in cAMP assays using Human PTH1R CHOK1 (FIG. 13A) andMouse PTH1R CHOK1 (FIG. 13B) cells. Schild regression analyses wereperformed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego,Calif.), revealing non-parallel rightward shifts inconcentration-response curves. These results are consistent with anon-competitive (allosteric) mechanism of action of the variant IgG.

FIGS. 14A-14B: Effects of PTH1R antibodies on PTH stimulated secretionof M-CSF by Saos-2 cells. Post confluent Saos-2 cells were treated for48 hr with the specified concentrations of PTH (1-34) peptide in thepresence of 40 μg/mL antibody. The clone XPA.85.017 (triangles) is shownalong with a light chain swapped variant XPA.85.287 (solid circles) andthe negative control IgG (open circles) (FIG. 14A). Five of the affinityenhanced light chain swapped variants containing the XPA.85.012 heavychain were shown to inhibit PTH stimulated M-CSF secretion (FIG. 14B).Control IgG (open circles), XPA.85.288 (solid circles), XPA.85.342(inverted triangles), XPA.85.346 (open squares), XPA.85.331 (opentriangles), and XPA.85.327 (solid squares).

FIGS. 15A-15C: Effects of PTH1R antibodies on PTHrP stimulated secretionof M-CSF by Saos-2 cells. Post confluent Saos-2 cells were treated for48 hr with the specified concentrations of PTHrP (1-36) peptide in thepresence of 40 μg/mL antibody. Negative control IgG (open circles),XPA.85.328 (inverted triangles), XPA.85.329 (solid circles), XPA.85.330(squares). Three of the affinity enhanced light chain swapped variantswith the XPA.85.012 heavy chain were shown to inhibit PTHrP stimulatedM-CSF secretion at a fixed antibody concentration of 40 m/mL (FIG. 15A).A fixed PTHrP (1-36) concentration of 12.5 nM was tested against avarious concentrations of the antibodies (FIG. 15B). In a separateexperiment, the parental clones XPA.85.012 and XPA.85.017 were comparedto their affinity matured variants XPA.85.288 and XPA.85.287respectively at 40 μg/mL (FIG. 15C).

FIGS. 16A-16B: Concentration-response curves for PTH (FIG. 16A) andPTHrP (FIG. 16B) peptide stimulation of hPTH1R mediated phosphorylationof ERK1/2 in the presence of 200 nM of antibody. CHO-hPTH1R cells wereincubated for 10 minutes at 37° C. with the antibodies followed by a 5minute incubation with increasing concentrations of peptide ligand, thenthe cells were lysed and the level of ERK1/2 phosphorylation wasdetermined by immuno-assay. Data points shown are mean values+/−SEM.Antibodies: Negative Control IgG (open circles), XPA.85.012 (solidcircles), and XPA.85.017 (solid triangles).

FIG. 17: PTH1R antibodies ability to reduce PTH(1-34)-induced elevationof serum calcium levels measured in vivo using aThyroparathyroidectomized (TPTx) model. Sprague-Dawley (SD) male rats(n=5-6/group) were challenged intravenously with PTH1R antibodiesXPA.85.017 (Ab017), XPA.85.287 (Ab287), XPA.85.288 (Ab288) and BM2, oran isotype control (15 mg/kg), 18 h before initiation of PTH(1-34)infusion. Serum calcium was measured before dosing (baseline),pre-infusion (T0), 2, 4 and 6 hr post start of infusion.

FIGS. 18A-18B: PTH1R antibodies ability to reduce PTH(1-34)-inducedelevation of serum calcium levels measured in vivo using a continuousinfusion model to mimic PTH hypersecretion in patients withhyperparathyroidism. FIG. 18A, hPTH(1-34) was continuously infusedsubcutaneously (Alzet mini pump, model 2ML1; 10 μl/hr, 10 mg/kg/day) innormal Sprague Dawley rats (Harlan) for 7 days to mimic PTHhypersecretion in patients with hyperparathyroidism. Calcium wasmeasured as a biomarker to assess in vivo neutralization by a singleintravenous administration of Ab288 (2 or 10 mg/kg; n=5/group), BM2 (10mg/kg; n=5), or isotype control (10 mg/kg, n=2) 24 hr after pumpimplantation. Serum calcium was measured before pump implantation(Predose), 24, 27 (3 hr post dose), 48, 72, 96, 120, 144 and 168 hr postpump implantation. FIG. 18B, antibodies, Ab328, Ab329 and Ab330 (2 mg/kgIV) along with Ab288 were tested in a similar study where serum calciumwas measured before pump implantation (Predose), 24, 27 (3 hr postdose), 48, 72, 96 and 120 hr post pump implantation. All antibodiessignificantly lowered levels of calcium 24 hr post dose and throughoutthe infusion, with Ab288 and Ab328 producing the most dramaticreduction.

FIGS. 19A-19B: PTH1R antibodies' ability to reduce PTHrP(1-34)-inducedelevation of serum calcium levels measured in vivo using a continuousinfusion model to mimic hypercalcemia in patients withhyperparathyroidism. The effects of anti-PTH1R receptor antibodies oninhibition of PTHrP activity were assessed by continuously infusinghPTHrP(1-36) subcutaneously (Alzet mini pump, model 2ML1; 10 μl/hr, 100mg/kg/day) in normal Sprague Dawley rats (Harlan) for 6 days to mimichypercalcemia in patients with hyperparathyroidism. FIG. 19A, calciumwas measured as a biomarker to assess in vivo neutralization by a singleintravenous administration of Ab288 (2 or 10 mg/kg; n=4/group),anti-PTHrP antibody MCB1.1 (10 mg/kg; n=5), or isotype control (10mg/kg, n=3) 24 hr after pump implantation. Serum calcium was measuredbefore pump implantation (Predose), 24, 26 (2 hr post dose), 48, 72, 96,120 and 144 hr post pump implantation. FIG. 19B, body weights weremeasured before pump implantation (Predose), 26 (2 hr post dose), 48, 96and 144 hr post pump implantation.

FIGS. 20A-20E: PTH1R antibody XPA.85.349 potently reduces mouse colon 26tumor-related hypercalcemia for a sustained period. Total serum calciumwas measured as a biomarker to assess in vivo neutralization by a singleintravenous administration of PTH1R antibody XPA.85.349 (2 mg/kg;n=6-8/group or 6 mg/kg; n=5-8/group), anti-KLH.G2 negative control forbinding (6 mg/kg; n=2-3), no treatment control (n=5), 24 hr after pumpimplantation (FIG. 20A shows mean serum total levels+/−S.E.M.). Micebody weight (FIG. 20B) and tumor weight (FIG. 20C) were measured overtime (48 hours prior to dosing and 48, 72 and 120 hours post dose).Levels of PTHrP (FIG. 20D) and PTH1-84 (FIG. 20E) were also measured inthe serum of mice 24, 48, 72 and 120 hours post dose with 2 or 6 mg/kgPTH1R antibody XPA.85.349, anti-KLH.G2 control antibody or prior totreatment and in animals without tumors.

FIG. 21: Sequence table for Heavy Chain (HC), Light Chain (LC)Complementarity-determining regions (CDRs) and variable regions. HC andLC CDRs and variable regions for XPA.85.332, XPA.85.012, XPA.85.345,XPA.85.329, XPA.85.326, XPA.85.328, XPA.85.288, XPA.85.342, XPA.85.333,XPA.85.343, XPA.85.327, XPA.85.330, XPA.85.334, XPA.85.344, XPA.85.346,XPA.85.331, XPA.85.347, XPA.85.017, XPA.85.341, XPA.85.335, XPA.85.340,XPA.85.339, XPA.85.287, XPA.85.336, XPA.85.337 and XPA.85.338 antibodiesand their respective Sequence Identifier (SEQ ID) numbers are provided.

DETAILED DESCRIPTION

The invention provides materials, methods, and uses relating to humanantibodies against PTH1R. In particular, the present disclosure providesmethods of use of such antibodies in the treatment of hypercalcemia,cancer, Humoral Hypercalcemia of Malignancy (HHM), PrimaryHyperparathyroidism (PHPT) and Secondary Hyperparathyroidism (SHPT) andcachexia.

The present disclosure provides molecules or agents that interact withparathyroid hormone receptor 1 (PTH1R) and inhibit or block one or morefunctional effects, such as for example signaling through bindingpartners, PTH and PTHrP. The present disclosure provides therapeuticsfor treating hypercalcemia, cancer, Humoral Hypercalcemia of Malignancy(HHM), or Primary Hyperparathyroidism (PHPT), SecondaryHyperparathyroidism (SHPT) and cachexia.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

“cAMP” as used herein, refers to cyclic adenosine mono-phosphate.

“CHO” as used herein, refers to Chinese hamster ovary cells.

“CHOK1” as used herein, refers to a subclone of the parental CHO cellline, which was derived from the ovary of an adult Chinese hamster.

“ERK1/2” as used herein, refers to extracellular signal-regulatedkinase.

“FLAG” as used herein, is a polypeptide protein tag that can be added toa recombinant PTH1R protein.

“HHM” as used herein, refers to humoral hypercalcemia of malignancy.

“MAPK” as used herein, refers to mitogen-activated protein kinase.

“PTH” as used herein, refers to parathyroid hormone.

“PTH1R” as used herein, refers to parathyroid hormone receptor 1. Alsoknown as PTHR1.

“PHPT” as used herein, refers to primary hyperparathyroidism.

“PTHrP” as used herein, refers to parathyroid hormone-related protein,and is also known as parathyroid hormone-like protein (PTHLP) orparathyroid hormone-like hormone (PTHLH).

An “immunoglobulin” or “native antibody” is a tetrameric glycoprotein.In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa (κ) and lambda (λ) light chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W.,ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light and heavy chain variabledomains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).

Immunoglobulin variable domains exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions or CDRs. From N-terminus to C-terminus, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al.,Nature 342:878-883, 1989.

The hypervariable region of an antibody refers to the CDR amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a CDR [e.g.,residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain as described by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)] and/or thoseresidues from a hypervariable loop (e.g., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917 (1987)]. CDRshave also been identified and numbered according to ImMunoGenTics (IMGT)numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc,M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), which describes theCDR locations in the light and heavy chain variable domains as follows:CDR1, approximately residues 27 to 38; CDR2, approximately residues 56to 65; and, CDR3, approximately residues 105 to 116 (germline) orresidues 105 to 117 (rearranged). In one embodiment, it is contemplatedthat the CDRs are located at approximately residues 26-31 (L1), 49-51(L2) and 88-98 (L3) in the light chain variable domain and approximatelyresidues 26-33 (H1), 50-58 (H2) and 97-111 (H3) in the heavy chainvariable domain of an antibody heavy or light chain of approximatelysimilar length to those disclosed herein. However, one of skill in theart understands that the actual location of the CDR residues may varyfrom the projected residues described above when the sequence of theparticular antibody is identified.

Framework or FR residues are those variable domain residues other thanthe hypervariable region residues.

“Heavy chain variable region” as used herein refers to the region of theantibody molecule comprising at least one complementarity determiningregion (CDR) of said antibody heavy chain variable domain. The heavychain variable region may contain one, two, or three CDR of saidantibody heavy chain.

“Light chain variable region” as used herein refers to the region of anantibody molecule, comprising at least one complementarity determiningregion (CDR) of said antibody light chain variable domain. The lightchain variable region may contain one, two, or three CDRs of saidantibody light chain, which may be either a kappa or lambda light chaindepending on the antibody.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments that can bind an antigen (e.g., Fab′,F′(ab)2, Fv, single chain antibodies, diabodies, Fcabs), and recombinantpeptides comprising the forgoing as long as they exhibit the desiredbiological activity. An “immunoglobulin” or “tetrameric antibody” is atetrameric glycoprotein that consists of two heavy chains and two lightchains, each comprising a variable region and a constant region.Antigen-binding portions may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies. Antibodyfragments or antigen-binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as one, two,three, four, five or six CDR sequences, as long as the antibody retainsthe desired biological activity.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

“Antibody variant” as used herein refers to an antibody polypeptidesequence that contains at least one amino acid substitution, deletion,or insertion in the variable region of the reference antibody variableregion domains. Variants may be substantially homologous orsubstantially identical to the unmodified antibody.

A “chimeric antibody,” as used herein, refers to an antibody containingsequence derived from two different antibodies (see, e.g., U.S. Pat. No.4,816,567) which typically originate from different species. Mosttypically, chimeric antibodies comprise human and rodent antibodyfragments, generally human constant and mouse variable regions.

A “neutralizing antibody” is an antibody molecule which is able toeliminate or significantly reduce a biological function of a targetantigen to which it binds. Accordingly, a “neutralizing” anti-targetantibody is capable of eliminating or significantly reducing abiological function, such as enzyme activity, ligand binding, orintracellular signaling.

An “allosteric antibody” or “an antibody that binds allosterically” anantibody that binds to a portion of PTH1R that is distinct from theactive ligand-binding site, i.e., is non-competitive with the naturalligands for the receptor. It is contemplated that an allosteric antibodydoes not appreciably change the binding affinity of PTH or PTHrP andPTH1R by more than 2-fold. In various embodiments, the antibody is anegative modulator that binds allosterically to PTH1R, optionally,wherein the antibody is capable of weakening the binding affinitybetween PTH or PTHrP and with PTH1R by at least about 2-fold, optionallyup to 1000-fold.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

As used herein, an antibody that “specifically binds” is “targetspecific”, is “specific for” target or is “immunoreactive” with thetarget antigen refers to an antibody or antibody substance that bindsthe target antigen with greater affinity than with similar antigens. Inone aspect of the disclosure, the target-binding polypeptides, orfragments, variants, or derivatives thereof, will bind with a greateraffinity to human target as compared to its binding affinity to targetof other, i.e., non-human, species, but binding polypeptides thatrecognize and bind orthologs of the target are within the scopeprovided.

For example, a polypeptide that is an antibody or fragment thereof“specific for” its cognate antigen indicates that the variable regionsof the antibodies recognize and bind the polypeptide of interest with adetectable preference (i.e., able to distinguish the polypeptide ofinterest from other known polypeptides of the same family, by virtue ofmeasurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween family members). It will be understood that specific antibodiesmay also interact with other proteins (for example, S. aureus protein Aor other antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody for use in the methods ofthe present disclosure are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies for use in themethods can be produced using any method known in the art.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a selective binding agent at one ormore of the antigen binding regions. Epitopes usually consist ofchemically active surface groupings of molecules, such as, amino acidsor carbohydrate side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.Epitopes as used herein may be contiguous or non-contiguous. Moreover,epitopes may be mimetic (mimotopes) in that they comprise a threedimensional structure that is identical to the epitope used to generatethe antibody, yet comprise none or only some of the amino acid residuesfound in the target that were used to stimulate the antibody immuneresponse. As used herein, a mimotope is not considered a differentantigen from the epitope bound by the selective binding agent; theselective binding agent recognizes the same three-dimensional structureof the epitope and mimotope.

The term “derivative” when used in connection with antibody substancesand polypeptides of the present disclosure refers to polypeptideschemically modified by such techniques as ubiquitination, conjugation totherapeutic or diagnostic agents, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as PEGylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins. Derivatives retain the bindingproperties of underivatized molecules of the disclosure.

The term “therapeutically effective amount” is used herein to indicatethe amount of target-specific composition of the disclosure that iseffective to ameliorate or lessen symptoms or signs of disease to betreated.

The terms “treat”, “treated”, “treating” and “treatment”, as used withrespect to methods herein refer to eliminating, reducing, suppressing orameliorating, either temporarily or permanently, either partially orcompletely, a clinical symptom, manifestation or progression of anevent, disease or condition. Such treating need not be absolute to beuseful.

The present methods provides for use of target-specific antibodies,which may comprise those exemplary sequences set out herein, fragments,variants and derivatives thereof, pharmaceutical formulations includingtarget-specific antibodies recited herein. Depending on the amino acidsequence of the constant domain of their heavy chains, immunoglobulinscan be assigned to different classes, IgA, IgD, IgE, IgG and IgM, whichmay be further divided into subclasses or isotypes, e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have ADCC activity. An antibody disclosed herein, ifit comprises a constant domain, may be of any of these subclasses orisotypes.

The antibodies used in the present methods may exhibit binding affinityto one or more PTH1R antigens of a K_(d) of less than or equal to about2×10⁻⁶ M, less than or equal to about 10⁻⁶ M, or less than or equal toabout 10⁻⁷ M, or less than or equal to about 10⁻⁸ M, or less than orequal to about 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less. In oneembodiment the antibodies have a K_(d) of at least 2×10⁶ M. Suchaffinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using surface plasmon resonance (SPR)technology (e.g., the BIAcore 2000 instrument, using general proceduresoutlined by the manufacturer); by radioimmunoassay using ¹²⁵I labeledtarget antigen; or by another method set forth in the examples below orknown to the skilled artisan. The affinity data may be analyzed, forexample, by the method of Scatchard et al., (Ann N.Y. Acad. Sci.,51:660, 1949).

A KinExA kinetic exclusion assay is also useful to measure the affinityof an antibody for its antigen. KinExA technology measures bindingevents in the solution phase, rather than binding events between asolution phase and a solid phase. In addition, while many methods formeasuring binding events require at least one reactant be modifiedthrough immobilization or labeling, the KinExA method does not requiremodification of molecules under study. The KinExA method is believed toallow a wider range of binding constants to be measured than othermethods currently available. Additional description about KinExA devicesand operation for antibody characterization is available from themanufacturer (Sapidyne Instruments, Inc., Boise, Id.) and can be foundin the published literature, for example U.S. Pat. No. 6,664,114 andDarling et al., “Kinetic Exclusion Assay Technology: Characterization ofMolecular Interactions.” Assay and Drug Development Technologies, 2004,2:647-657.

Parathyroid Hormone Receptor 1

Parathyroid hormone receptor 1 (PTH1R) is composed of an N-terminalextracellular domain (N-ECD), seven TM helices, three ECs, threeintracellular loops (ICs), and a C-terminal intracellular domain (C-ICD)(Thomas et al., 2008 J Bone Miner Res. 24(5): 925-934). This protein isa receptor for parathyroid hormone (PTH) and for parathyroid hormonerelated peptide (PTHrP). The activity of PTH1R is mediated by G proteinswhich activate adenylyl cyclase and also a phosphatidylinositol-calciumsecond messenger system. PTH1R is highly expressed in bone, kidney andgrowth plates, and in other tissues is expressed at lower levels atvarious times throughout development (e.g. skeleton, heart, and mammaryglands) (Cheloha et al., 2015 Nature Reviews Endocrinology 11, 712-724;Shimizu et al., 2004 JBC 280(3):1797-807). Both elevated PTH and PTHrPcause hypercalcemia resulting in symptoms of stones (kidney stones),bone loss (bone resorption), and psychotic overtones (depression,anxiety, cognitive dysfunction, insomnia, coma). In cancer patients,elevated PTHrP levels lead to increased osteoclastic bone resorption andhypercalcemia, a condition known as humoral hypercalcemia of malignancy(Cheloha et al., 2015 Nature Reviews Endocrinology 11, 712-724).

Humoral Hypercalcemia of Malignancy

Humoral Hypercalcemia of Malignancy (HHM) results from excessiveproduction of PTHrP by certain tumors (Hoare et al., 2002 Peptides 23:989-998). HHM is a very common complication of particular cancers suchas breast cancer and multiple myeloma, and lung carcinoma (Findlay etal., 1980 Cancer Research 40, 1311-1317). In 2012, 2.7% of cancerpatients had HHM in the US (solid tumors and hematologicalmalignancies).

Primary Hyperparathyroidism

Disorders of the parathyroid glands include hyperparathyroidism andhypoparathyroidism. Primary Hyperparathyroidism (PHPT) occurs when theprimary defect is in the parathyroid gland itself, resulting in releaseof excess PTH (Shimizu et al., 2005 JBC 280(3):1797-807). PHPT canresult in dysbolism of calcium, thereby causing disorders such ashypercalcemia, hypophosphatemia, osteitis fibrosa, nephrolithiasis, andhypertension. PHPT is also the most common cause of hypercalcemia(Marcocci et al., 2011 NEJM 365:2389-2397). Excessive circulating levelsof PTH, as occurs in cases of hyperparathyroidism, or PTHrP, asfrequently occurs in cancer because of secretion by malignant tumors,produces a hypercalcemic state, which can be severely debilitating andpotentially fatal (Shimizu et al., 2005 JBC 280(3):1797-807).

Therapy for hyperparathyroidism is usually directed at surgical removalof the offending parathyroid tissue. Patients with HHM usually cannot becured surgically, and must be managed medically usually directed atpreventing bone resorption (bisphosphonates or calcitonin) or promotingcalcium excretion by the kidneys (saline diuresis) with variableeffectiveness (Rosen et al. Calcif. Tissue Int. 61, 455-459).

PTH1R Inhibitors

PTH1R antagonists have been shown to be of benefit in cases of HHM andhyperparathyroidism (Dresner-Pollak et al. 1996 J. Bone Miner. Res. 11,1061-1065; Cheloha et al., 2015 Nature Reviews Endocrinology 11,712-724). The development of small-molecule ligands that mimic theactions of the agonist peptides has been a challenging goal for thePTH1R. Several inhibitors of PTH1R for use in treating HHM and PHPT areknown in the art (U.S. Pat. No. 7,150,974; U.S. Pat. No. 7,985,835; U.S.Pat. No. 7,910,544).

PTH1R Antibodies

In various embodiments, the disclosure provides an antibody specific forPTH1R with an affinity K_(d) of 10⁻⁶ M or less. In various embodiments,the disclosure provides an antibody specific for PTH1R with an affinityK_(d) of 2×10⁻⁶M or less. In exemplary embodiments, an anti-PTH1Rantibody described herein binds at least with an affinity of 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or less. In other embodiments, an antibodydescribed herein binds to PTH1R with at least 2-50 fold, 10-100 fold,2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%,50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higheraffinity (e.g., preferentially binds to PTH1R) compared to binding toPTH2R. In certain embodiments, the affinity is measured by surfaceplasmon resonance or KinExA assay.

In a related aspect, the antibody binds the N-terminal portion of PTH1R.In a further aspect, the disclosure contemplates an antibody that doesnot bind parathyroid hormone receptor 2 (PTH2R). In various aspects theantibody binds PTH1R on the surface of a cell. In certain aspects theantibody binds allosterically to PTH1R.

In a preferred embodiment, the antibody is a negative modulatorantibody, optionally wherein the antibody is capable of weakening thebinding affinity between PTH or PTHrP and with PTH1R by at least about2-fold, optionally up to 1000-fold. In other embodiments, an antibodydescribed herein is capable of weakening the binding affinity betweenPTH or PTHrP with at least 2-1000 fold, 10-100 fold, 2-fold, 5-fold,10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold,500-fold, 600-fold, 700-fold, 800-fold, 900-fold or 1000-fold.

In a various embodiments, the antibody inhibits calcium flux in a cellin response to stimulation of the receptor with parathyroid hormone(PTH) or parathyroid hormone related protein (PTHrP). In relatedembodiments, the antibody inhibits PTH- or PTHrP-mediated cyclicadenosine mono-phosphate (cAMP) accumulation.

In one embodiment, the PTH1R antibody is a monoclonal antibody.

In one aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising (a) a heavy chaincomplementary determining repeat (CDR)1 amino acid sequence set forth inFIG. 21 or SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60,63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variantthereof in which one or two amino acids have been changed; (b) a heavychain CDR2 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 28,31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82,85, 88, 91, 94, 97, 100, 103 that is from the same heavy chain variableregion as (a), or a variant thereof in which one or two amino acids havebeen changed; and (c) a heavy chain CDR3 amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59,62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that isfrom the same heavy chain variable region as (a), or a variant thereofin which one or two amino acids have been changed.

In a related aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising: (a) a heavy chainCDR1 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 27, 30, 33,36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87,90, 93, 96, 99, 102, or a variant thereof having at least 70% identitythereto; (b) a heavy chain CDR2 amino acid sequence set forth in SEQ IDNOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76,79, 82, 85, 88, 91, 94, 97, 100, 103 that is from the same heavy chainvariable region as (a), or a variant thereof having at least 70%identity thereto; and (c) a heavy chain CDR3 amino acid sequence setforth in SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65,68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that is from thesame heavy chain variable region as (a), or a variant thereof having atleast 70% identity thereto.

In a further aspect, the disclosure contemplates an antibody that bindsan antibody that binds parathyroid hormone receptor 1 (PTH1R)comprising: (a) a heavy chain CDR1 amino acid sequence set forth in SEQID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72,75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variant thereof having atleast 70% identity thereto; (b) an independently selected heavy chainCDR2 amino acid sequence set forth in in FIG. 21 or SEQ ID NOs: 28, 31,34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85,88, 91, 94, 97, 100, 103, or a variant thereof having at least 70%identity thereto; and (c) an independently selected heavy chain CDR3amino acid sequence set forth in in FIG. 21 or SEQ ID NOs: 29, 32, 35,38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89,92, 95, 98, 101, 104, or a variant thereof having at least 70% identitythereto.

In certain embodiments, at least two of the heavy chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:27-104. In a related embodiment, three of the heavy chain CDR1, CDR2 andCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:27-104.

In some embodiments, an antibody contemplated herein comprises an aminoacid sequence at least 85% identical to a heavy chain variable regionamino acid sequence set forth in FIG. 21 or SEQ ID NOs: 1-26. In someembodiments, provided herein is an antibody that comprises an amino acidsequence at least 95% identical to a heavy chain variable region aminoacid sequence set forth in FIG. 21 or SEQ ID NOs: 1-26.

It is further contemplated that an antibody described herein comprises apolypeptide sequence having an amino acid sequence at least 70%identical over all three HCDRs in a heavy chain variable region, theamino acid sequences of HCDR1, HCDR2 and HCDR3 set forth in FIG. 21 orSEQ ID NOs: 27-104.

In another embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to all three HCDRs in theheavy chain variable region of an antibody sequence in FIG. 21 or theCDRs set out in SEQ ID NOs: 27-104.

In a related embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to all three LCDRs in thelight chain variable region of an antibody sequence in FIG. 21 or theCDRs set out in SEQ ID NOs: 131-208.

In a further embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to all six CDRs in theheavy chain and light chain variable regions of an antibody sequence inFIG. 21 or the CDRs set out in SEQ ID NOs: 27-104 and 131-208.

In certain embodiments, an antibody contemplated herein is one in whichone or more heavy chain framework amino acids have been replaced withcorresponding amino acid(s) from another human antibody amino acidsequence.

In one embodiment, an antibody contemplated herein further comprises anyone of the light chain CDR amino acid sequences set forth in FIG. 21 orSEQ ID NOs: 131-208. In some embodiments, an antibody comprises at leasttwo of the light chain CDR amino acid sequences set forth in FIG. 21 orSEQ ID NOs: 131-208. In other embodiments, an antibody comprises atleast three of the light chain CDR amino acid sequences set forth inFIG. 21 or SEQ ID NOs: 131-208.

In another aspect, an antibody described herein comprises (a) a lightchain CDR1 amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 131,134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173,176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or a variantthereof in which one or two amino acids have been changed, or aconsensus sequence thereof set out in FIG. 21; (b) a light chain CDR2amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 132, 135, 138,141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180,183, 186, 189, 192, 195, 198, 201, 204, 207 that is from the same lightchain variable region as (a), or a variant thereof in which one or twoamino acids have been changed, or a consensus sequence thereof set outin FIG. 21; and (c) a light chain CDR3 amino acid sequence set forth inFIG. 21 or SEQ ID NOs: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160,163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202,205, 208 that is from the same light chain variable region as (a), or avariant thereof in which one or two amino acids have been changed, or aconsensus sequence thereof set out in FIG. 21.

In alternative embodiments, an antibody contemplated herein comprises:(a) a light chain CDR1 amino acid sequence set forth in FIG. 21 or SEQID NOs: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167,170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or avariant thereof in which one or two amino acids have been changed; (b)an independently selected light chain CDR2 amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 132, 135, 138, 141, 144, 147, 150, 153, 156,159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198,201, 204, 207, or a variant thereof in which one or two amino acids havebeen changed; and (c) an independently selected light chain CDR3 aminoacid sequence set forth in FIG. 21 or SEQ ID NOs: 133, 136, 139, 142,145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184,187, 190, 193, 196, 199, 202, 205, 208, or a variant thereof in whichone or two amino acids have been changed.

In certain embodiments, at least two of the light chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in FIG. 21 or SEQ ID NOs:131-208.

It is further contemplated that an antibody described herein comprisesan amino acid sequence at least 70% identical to a light chain variableregion amino acid sequence set forth in FIG. 21 or SEQ ID NOs: 105-130.In a related embodiment, the antibody comprises an amino acid sequenceat least 85% identical to a light chain variable region amino acidsequence set forth in FIG. 21 or SEQ ID NOs: 105-130. In a furtherembodiment, the antibody comprises an amino acid sequence at least 95%identical to a light chain variable region amino acid sequence set forthin FIG. 21 or SEQ ID NOs: 105-130. In still another embodiment, theantibody comprises a light chain variable region amino acid sequence setforth in FIG. 21 or SEQ ID NOs: 105-130.

In a further embodiment, an antibody described herein comprises apolypeptide sequence having an amino acid sequence at least 70%identical over all three LCDRs of a light chain variable region, theamino acid sequences of LCDR1, LCDR2 and LCDR3 set forth in FIG. 21 orSEQ ID NOs: 131-208.

In certain embodiments, an antibody described herein comprises (i) anamino acid sequence at least 70% identical over all three LCDRs, of alight chain variable region, the amino acid sequences of LCDR1, LCDR2and LCDR3 set forth in FIG. 21 or SEQ ID NOs: 131-208 and (ii) an aminoacid sequence at least 70% identical over all three HCDRs of a heavychain variable region, the amino acid sequences of HCDR1, HCDR2 andHCDR3 set forth in FIG. 21 or SEQ ID NOs: 27-104.

In another aspect, the disclosure provides an antibody that bindsparathyroid hormone receptor 1 (PTH1R) comprising a light chain variableregion and/or a heavy chain variable region, wherein (a) the light chainvariable region comprises at least a CDR1 selected from SEQ ID NOs: 131,134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173,176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 or sequences atleast 80% identical thereto, a CDR2 selected from SEQ ID NOs: 132, 135,138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177,180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or sequences at least80% identical thereto, and/or a CDR3 selected from SEQ ID NOs: 133, 136,139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178,181, 184, 187, 190, 193, 196, 199, 202, 205, 208 or sequences at least80% identical thereto; and/or wherein (b) the heavy chain variableregion comprises at least a CDR1 selected from SEQ ID NOs: 27, 30, 33,36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87,90, 93, 96, 99, 102 or sequences at least 80% identical thereto, a CDR2selected from SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58,61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71,74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 80%identical thereto.

In a related embodiment, an antibody described herein comprises (a) alight chain variable region comprising at least a CDR1 selected from SEQID NO: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167,170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168,171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 orsequences at least 90% identical thereto, and a CDR3 selected from SEQID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169,172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 orsequences at least 90% identical thereto; and/or wherein (b) the heavychain variable region comprises at least a CDR1 selected from SEQ ID NO:27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78,81, 84, 87, 90, 93, 96, 99, 102 or sequences at least 90% identicalthereto, a CDR2 selected from SEQ ID NO: 28, 31, 34, 37, 40, 43, 46, 49,52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103or sequences at least 90% identical thereto, and a CDR3 selected fromSEQ ID NO: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71,74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 90%identical thereto.

In some embodiments, an antibody of the disclosure further comprises aheavy chain constant region, wherein the heavy chain constant region isa modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, orcombinations thereof.

In certain embodiments, an antibody is provided in which one or morelight chain framework amino acids have been replaced with correspondingamino acid(s) from another human antibody amino acid sequence,optionally wherein the framework comprises one or more of the changesset out in FIGS. 1A and 1B.

In one aspect, the antibody of the disclosure is selected from the groupconsisting of XPA.85.012, XPA.85.017, XPA.85.288, XPA.85.328,XPA.85.329, XPA.85.330 and XPA.85.349.

In one embodiment, an antibody described herein further comprises ahuman light chain constant region attached to said light chain variableregion. In some embodiments, the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof.

In another aspect, the disclosure provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the heavy chainor light chain as described herein. In various embodiments thenucleotide sequences encoding the antibody variable regions are set outin SEQ ID NOs: 209-234 (heavy chain) and 235-260 (light chain).

In various embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the light chain variable region amino acidsequence of any one of SEQ ID NOs: 105-130 or a fragment thereof. In oneembodiment, the nucleic acid molecule comprises the light chain variableregion nucleotide sequence of any one of SEQ ID NOs: 235-260, or afragment thereof. In further embodiments, the nucleic acid moleculecomprises a nucleotide sequence that encodes the heavy chain variableregion amino acid sequence of any one of SEQ ID NOs: 1-26 or a fragmentthereof. In one embodiment, the nucleic acid molecule comprises theheavy chain variable region nucleotide sequence of any one of SEQ IDNOs: 209-234, or a fragment thereof. Nucleic acid molecules of thedisclosure further include all nucleic acid sequences, including thesequences in SEQ ID NOs: 209-260, and nucleic acid sequences comprisesdegenerate codons based on the diversity of the genetic code, encodingan amino acid sequence of the heavy and light chain variable regions ofan antibody described herein or any HCDRs or LCDRs described herein, andencoding the CDR amino acid sequences as set out in SEQ ID NOs: 27-104and 131-208, as well as nucleic acids that hybridize under highlystringent conditions, such as those described herein, to a nucleic acidsequence encoding an amino acid sequence of the heavy and light chainvariable regions of an antibody described herein set out in SEQ ID NO:1-26 or 105-130 or any HCDRs or LCDRs described herein as set out in SEQID NOs: 27-104 or 131-208.

In some embodiments, the nucleic acid molecule encodes a VL amino acidsequence that is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96 97, 98 or 99% identical to a VL amino acid sequence set out inSEQ ID NOs: 105-130. In a related aspect, the VL amino acid sequence isa consensus sequence. Nucleic acid molecules of the disclosure includenucleic acids that hybridize under highly stringent conditions, such asthose described herein, to a nucleic acid sequence encoding the lightchain variable region amino acid sequence of SEQ ID NOs: 105-130, orthat has the light chain variable region nucleic acid sequence of SEQ IDNOs: 235-260. In some embodiments, the nucleic acid encodes the aminoacid sequence of the heavy chain CDRs of said antibody set out in SEQ IDNO: 131-208.

It is further contemplated that a nucleic acid molecule of thedisclosure comprises a nucleotide sequence that encodes the VH aminoacid sequence of any one of antibodies described herein, or a fragmentthereof. In some embodiments, the nucleic acid encodes the amino acidsequence of the heavy chain and/or light chain CDRs of said antibody. Insome embodiments, said fragment is a contiguous fragment comprisingheavy chain and/or light chain CDR1-CDR3. In one embodiment, saidfragment comprises at least one, two or three of a heavy chain and/orlight chain CDR1, CDR2, or CDR3 region, optionally with a differenthuman or human consensus framework, and optionally with 1, or up to 2,or up to 3 mutations in the CDRs. CDR amino acid sequences are set outin SEQ ID NOs: 27-104 and 131-208.

In a related aspect, the nucleic acid molecule comprises a nucleotidesequence that encodes the heavy chain variable region amino acidsequence of one of heavy chain of SEQ ID NOs: 1-26, or a fragmentthereof. In one embodiment, the nucleic acid molecule comprises theheavy chain variable region having a nucleotide sequence set out in SEQID NOs: 209-234, or a fragment thereof.

In some embodiments, the nucleic acid molecule encodes a VH amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a VH amino acid sequence set out in SEQ ID NOs: 1-26.In a related aspect, the VH amino acid sequence is a consensus sequence.Nucleic acid molecules of the disclosure further include nucleic acidsthat hybridize under highly stringent conditions, such as thosedescribed herein, to a nucleic acid sequence encoding the heavy chainvariable region amino acid sequence of SEQ ID NOs: 1-26, or that has theheavy chain variable region nucleic acid sequence of any one of SEQ IDNOs: 209-234.

It is further contemplated that the nucleic acids of the disclosure mayencode a full-length light chain or heavy chain of an antibody selectedfrom the antibodies set out in FIG. 21 wherein a full-length light chainor full-length heavy chain comprises a light chain constant region or aheavy chain constant region, respectively, light chain constant regionsoptionally include unmodified or modified kappa or lambda regions, andheavy constant regions include unmodified or modified constant regionsof any of the classes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, orIgE.

In one aspect, the full length variable light chain antibody comprisesthe amino acid sequences set out in SEQ ID NOs: 105-130. It is furthercontemplated that the nucleotide sequence encoding the full-length lightchain encodes the amino acid sequences set out in SEQ ID NOs: 105-130and comprises the nucleotide sequences set forth in SEQ ID NOs: 235-260.

In one aspect, the full length variable heavy chain antibody comprisesthe sequences in any one of SEQ ID NOs: 1-26. Further provided arenucleotide sequences that encode the full-length heavy chain variableregion amino acid sequences set out in SEQ ID NOs: 1-26, and comprisethe nucleotide sequences set forth in any one of SEQ ID NOs: 209-234.

It is further contemplated that antibodies of the present disclosure maybe used as smaller antigen binding fragments of the antibody that arewell-known in the art and described herein, as well as derivatives andmodified antibodies as described herein.

Monoclonal Antibodies

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against the same ordifferent determinants (epitopes). In addition to their specificity,monoclonal antibodies are advantageous in that they are synthesized bythe homogeneous culture, uncontaminated by other immunoglobulins withdifferent specificities and characteristics.

As described below, antibodies, including monoclonal, humanized, andother antibodies described herein, contemplated herein are typicallygenerated recombinantly or through other methods of manipulating thegenetic code in vitro or in vivo, and are therefore not necessarilyreflective of a particular antibody that is found in nature.

Monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al. (Nature, 256:495-7, 1975) (Harlow & Lane;Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press:Cold Spring Harbor, N.Y. (1988); Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986), or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The monoclonal antibodies may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol.222:581-597, 1991). Additional methods for producing monoclonalantibodies are well-known to a person of ordinary skill in the art.

Monoclonal antibodies, such as those produced by the above methods, aresuitably separated from culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydrophobic interaction chromatography(HIC), ion exchange chromatography, hydroxyapatite chromatography, gelelectrophoresis, dialysis, and/or affinity chromatography.

It is further contemplated that antibodies of the present disclosure maybe used as smaller antigen binding fragments of the antibody that arewell-known in the art and described herein.

Antibody Fragments

Antibody fragments comprise a portion of an intact full length antibody,preferably an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fcab, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecfic,trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies);minibody; chelating recombinant antibody; tribodies or bibodies;intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP),binding-domain immunoglobulin fusion proteins; camelized antibodies; VHHcontaining antibodies; and other polypeptides formed from antibodyfragments. See for example Holliger & Hudson, 2005 Nat. Biotech.23:1126-36; Eyer & Hruska, Veterinarni Medicina 57: 439-513, 2012.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains each with a single antigen-binding site,and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, that has two “Single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of antibody, whereinthese domains are present in a single polypeptide chain. Preferably, theFv polypeptide further comprises a polypeptide linker between the VH andVL domains that enables the Fv to form the desired structure for antigenbinding, resulting in a single-chain antibody (scFv), in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988). For a review of scFv see Pluckthun, in ThePharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragmentconsists of the VH and CH1 domains.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a VH domain.Diabodies are bivalent antibodies in which VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g., EP404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVHH domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H2L2(referred to as “heavy-chain antibodies” or “HCAbs”). Camelid VHHreportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H2L2) antibodyisotype in which VH recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Camelid VHH domains have been found to bind to antigenwith high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001)and possess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Classical VH-only fragments are difficult to producein soluble form, but improvements in solubility and specific binding canbe obtained when framework residues are altered to be more VHH-like.(See, e.g., Reichman, et al., J Immunol Methods 1999, 231:25-38).Methods for generating antibodies having camelid heavy chains aredescribed in, for example, in U.S. Patent Publication Nos. 20050136049and 20050037421.

The variable domain of an antibody heavy-chain, a fully functionalantigen-binding fragment with a molecular mass of only 15 kDa, isreferred to as a nanobody (Cortez-Retamozo et al., Cancer Research64:2853-57, 2004). A nanobody library may be generated from an immunizeddromedary as described in Conrath et al., (Antimicrob Agents Chemother45: 2807-12, 2001) or using recombinant methods as described in Revetset al, Expert Opin. Biol. Ther. 5(1):111-24 (2005).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. AdditionalFab-based bispecific formats are described in Wu et al., mAbs 7:470-482, 2015.

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 17(4):315-23, 2004.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domain (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

Thus, a variety of compositions comprising one, two, and/or three CDRs(e.g., a single CDR alone or in tandem, 2, 3, or other multiple repeatsof the CDRs, or combinations of 2 or 3 CDRs alone or in tandem repeats;optionally, with a spacer amino acid sequence between the CDRs orrepeats) of a heavy chain variable region or a light chain variableregion of an antibody may be generated by techniques known in the art.

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental non-human (e.g., mouse) monoclonal antibodies, theycan be used for the treatment of humans with far less risk ofanaphylaxis.

Chimeric monoclonal antibodies, in which the variable Ig domains of anon-human (e.g., mouse) monoclonal antibody are fused to human constantIg domains, can be generated using standard procedures known in the art(See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984);and, Boulianne et al, Nature 312, 643-646, (1984)).

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”), or, alternatively, (3)substituting human amino acids at positions determined to be unlikely toadversely affect either antigen binding or protein folding, but likelyto reduce immunogenicity in a human environment (e.g., HUMANENGINEERING™). In the present disclosure, humanized antibodies willinclude both “humanized,” “veneered” and “HUMAN ENGINEERED™” antibodies.These methods are disclosed in, e.g., Jones et al., Nature 321:522 525(1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855(1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immunol. 31:169-217 (1994); Studnicka et al. U.S.Pat. No. 5,766,886; Studnicka et al., (Protein Engineering 7: 805-814,1994; Co et al., J. Immunol. 152, 2968-2976 (1994); Riechmann, et al.,Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng.4:773-783 (1991) each of which is incorporated herein by reference. CDRgrafting techniques are known in the field, see for example, Riechmann,et al. 1988 Nature 332:323-27). Additional antibody humanization methodsare reviewed by Safdan et al., Biotech. Gen. Eng. Rev. 29: 175-86, 2013.

Human Antibodies from Transgenic Animals

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268; Green L L,Curr Drug Discovery Technol., 11(1), 74-84, 2014; Lee E C et al., NatureBiotechnology, 32:356-363, 2014; Lee E C and Owen M, Methods Mol Biol.,901:137-48, 2012).

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), whichcontains gene sequences from unrearranged human antibody genes that codefor the heavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102 (2002)) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TCMOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S. Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human Antibodies from Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. Antibodies produced by phagetechnology are produced as antigen binding fragments—usually Fv or Fabfragments—in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered, for example, into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

By way of example, one method for preparing the library of antibodiesfor use in phage display techniques comprises the steps of immunizing anon-human animal comprising human immunoglobulin loci with targetantigen or an antigenic portion thereof to create an immune response,extracting antibody producing cells from the immunized animal; isolatingRNA from the extracted cells, reverse transcribing the RNA to producecDNA, amplifying the cDNA using a primer, and inserting the cDNA into aphage display vector such that antibodies are expressed on the phage.Recombinant target-specific antibodies of the disclosure may be obtainedin this way.

In another example, antibody producing cells can be extracted fromnon-immunized animals, RNA isolated from the extracted cells and reversetranscribed to produce cDNA, which is amplified using a primer, andinserted into a phage display vector such that antibodies are expressedon the phage. Phage-display processes mimic immune selection through thedisplay of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice. One such technique is described in WO 99/10494, whichdescribes the isolation of high affinity and functional agonisticantibodies for MPL and msk receptors using such an approach. Antibodiesof the disclosure can be isolated by screening of a recombinantcombinatorial antibody library, for example a scFv or Fab phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derivedfrom human lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. See e.g., U.S. Pat. No. 5,969,108. Thereare commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982, and Omidfar & Daneshpour, Exp. Op. Drug Disc. 10: 651-669,2015.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human VH and VL library arescreened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method may be scFvlibraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J 12:725-734(1993)). The antibody libraries preferably are screened using targetprotein as the antigen.

Alternatively, the Fd fragment (VH-CH1) and light chain (VL-CL) ofantibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. Through severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, Ike, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairings of selected VL and VH segmentsare screened for target binding, to select preferred VL/VH paircombinations (See, for example, Kang et al., Proc. Natl. Acad. Sci. 88:11120-11123, 1991). Additionally, to further improve the quality of theantibody, the VL and VH segments of the preferred VL/VH pair(s) can berandomly mutated, preferably within the any of the CDR1, CDR2 or CDR3region of VH and/or VL, in a process analogous to the in vivo somaticmutation process responsible for affinity maturation of antibodiesduring a natural immune response. This in vitro affinity maturation canbe accomplished by amplifying VL and VH regions using PCR primerscomplementary to the VH CDR1, CDR2, and CDR3, or VL CDR1, CDR2, andCDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode VL and VH segments into which randommutations have been introduced into the VH and/or VL CDR3 regions. Theserandomly mutated VL and VH segments can be rescreened for binding totarget antigen.

Following screening and isolation of a target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the disclosure, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783 (1992)).

Methods for display of peptides on the surface of yeast, microbial andmammalian cells have also been used to identify antigen specificantibodies. See, for example, U.S. Pat. Nos. 5,348,867; 5,723,287;6,699,658; Wittrup, Curr Op. Biotech. 12:395-99 (2001); Lee et al.,Trends in Biotech. 21(1) 45-52 (2003); Surgeeva et al, Adv. Drug Deliv.Rev. 58: 1622-54 (2006). Antibody libraries may be attached to yeastproteins, such as agglutinin, effectively mimicking the cell surfacedisplay of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingin vitro display methods and microbial cell display, including ribosomedisplay and mRNA display (Amstutz et al, Curr. Op. Biotech. 12: 400-05(2001)). Selection of polypeptides using ribosome display is describedin Hanes et al., (Proc. Natl Acad Sci USA, 94:4937-4942 (1997)) and U.S.Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome displayis also useful for rapid large scale mutational analysis of antibodies.The selective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

Amino Acid Sequence Variants

Modified polypeptide compositions comprising one, two, three, four,five, and/or six CDRs of an antibody may be generated, wherein a CDR isaltered to provide increased specificity or affinity to the targetmolecule. Sites within antibody CDRs are typically modified in series,e.g., by substituting first with conservative choices (e.g., hydrophobicamino acid substituted for a non-identical hydrophobic amino acid) andthen with more dissimilar choices (e.g., hydrophobic amino acidsubstituted for a charged amino acid), and then deletions or insertionsmay be made at the target site. For example, using the conservedframework sequences surrounding the CDRs, PCR primers complementary tothese consensus sequences are generated to amplify the antigen-specificCDR sequence located between the primer regions. Techniques for cloningand expressing nucleotide and polypeptide sequences are well-establishedin the art [see e.g. Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor, N.Y. (1989)]. The amplified CDRsequences are ligated into an appropriate plasmid. The plasmidcomprising one, two, three, four, five and/or six cloned CDRs optionallycontains additional polypeptide encoding regions linked to the CDR.

Modifications may be made by conservative or non-conservative amino acidsubstitutions described in greater detail below. “Insertions” or“deletions” are preferably in the range of about 1 to 20 amino acids,more preferably 1 to 10 amino acids. The variation may be introduced bysystematically making substitutions of amino acids in an antibodypolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity. Nucleic acid alterationscan be made at sites that differ in the nucleic acids from differentspecies (variable positions) or in highly conserved regions (constantregions). Methods for altering antibody sequences and expressingantibody polypeptide compositions useful in the disclosure are describedin the art. See e.g., U.S. Pat. No. 8,569,462

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions involve replacing anamino acid with another member of its class. Non-conservativesubstitutions involve replacing a member of one of these classes with amember of another class.

Conservative amino acid substitutions are made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline(Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine(Met, M); polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y),asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic)amino acids include arginine (Arg, R), lysine (Lys, K), and histidine(His, H); and negatively charged (acidic) amino acids include asparticacid (Asp, D) and glutamic acid (Glu, E).

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Altered Glycosylation

Antibody variants can also be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example,deleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

Fc glycans influence the binding of IgG to Fc receptors and C1q, and aretherefore important for IgG effector functions. Antibody variants withmodified Fc glycans and altered effector function may be produced. Forexample, antibodies with modified terminal sugars such as sialic acids,core fucose, bisecting N-acetylglucosamine, and mannose residues mayhave altered binding to the FcγRIIIa receptor and altered ADCC activity.In a further example, antibodies with modified terminal galactoseresidues may have altered binding to C1q and altered CDC activity (Raju,Curr. Opin. Immunol. 20:471-78, 2008).

Also contemplated for use in the methods are antibody molecules withabsent or reduced fucosylation that exhibit improved ADCC activity. Avariety of ways are known in the art to accomplish this. For example,ADCC effector activity is mediated by binding of the antibody moleculeto the FcγRIII receptor, which has been shown to be dependent on thecarbohydrate structure of the N-linked glycosylation at the Asn-297 ofthe CH2 domain. Non-fucosylated antibodies bind this receptor withincreased affinity and trigger FcγRIII-mediated effector functions moreefficiently than native, fucosylated antibodies. For example,recombinant production of non-fucosylated antibody in CHO cells in whichthe alpha-1,6-fucosyl transferase enzyme has been knocked out results inantibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al.,Biotechnol Bioeng. 87:614-22 (2004)). Similar effects can beaccomplished through decreasing the activity of this or other enzymes inthe fucosylation pathway, e.g., through siRNA or antisense RNAtreatment, engineering cell lines to knockout the enzyme(s), orculturing with selective glycosylation inhibitors (Rothman et al., MolImmunol. 26:1113-23 (1989)). Some host cell strains, e.g. Lec13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. (Shields et al., J Biol Chem. 277:26733-40 (2002);Shinkawa et al., J Biol Chem. 278:3466-73 (2003)). An increase in thelevel of bisected carbohydrate, e.g. through recombinantly producingantibody in cells that overexpress GnTIII enzyme, has also beendetermined to increase ADCC activity (Umana et al., Nat Biotechnol.17:176-80 (1999)). It has been predicted that the absence of only one ofthe two fucose residues may be sufficient to increase ADCC activity(Ferrara et al., Biotechnol Bioeng. 93:851-61 (2006)). Glycosylation ofantibodies and methods are reviewed in Niewa and Satoh, J.Pharmaceutical Sciences 104:930-41, 2015.

Variants with Altered Effector Function

Other modifications of the antibodies for use in the methods arecontemplated. In one aspect, it may be desirable to modify an antibodyused herein with respect to effector function, for example, to enhancethe effectiveness of the antibody in treating cancer. One method formodifying effector function teaches that cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., (J. Exp Med. 176: 1191-1195(1992)) and Shopes, B. (J. Immunol. 148: 2918-2922 (1992)). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., (CancerResearch 53: 2560-2565 (1993)). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,(Anti-Cancer Drug Design 3: 219-230 (1989)). In addition, it has beenshown that sequences within the CDR can cause an antibody to bind to MHCClass II and trigger an unwanted helper T-cell response. A conservativesubstitution can allow the antibody to retain binding activity yet loseits ability to trigger an unwanted T-cell response.

In certain embodiments of the present disclosure, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetherapeutic efficacy, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half-life,for example, adding molecules such as PEG or other water solublepolymers, including polysaccharide polymers, to antibody fragments toincrease the half-life.

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the CL region or VLregion, or both, of the antibody fragment.

Thus, antibodies of the present disclosure may comprise a human Fcportion, a human consensus Fc portion, or a variant thereof that retainsthe ability to interact with the Fc salvage receptor, including variantsin which cysteines involved in disulfide bonding are modified orremoved, and/or in which the a methionine is added at the N-terminusand/or one or more of the N-terminal 20 amino acids are removed, and/orregions that interact with complement, such as the C1q binding site, areremoved, and/or the ADCC site is removed [see, e.g., Sarmay et al.,Molec. Immunol. 29:633-9 (1992)].

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Variants thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA See also Presta et al., (Biochem. Soc. Trans.30:487-490, 2001), incorporated herein by reference in its entirety,which described several positions in the Fc region of IgG1 were foundwhich improved binding only to specific Fc gamma receptors (R) orsimultaneously improved binding to one type of Fc gamma R and reducedbinding to another type. Selected IgG1 variants with improved binding toFc gamma RIIIa were then tested in an in vitro antibody-dependentcellular cytotoxicity (ADCC) assay and showed an enhancement in ADCCwhen either peripheral blood mononuclear cells or natural killer cellswere used.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes variants with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedC1q binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcRII, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that Clq binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

U.S. Patent Publication No. 20040132101, incorporated by referenceherein in its entirety, describes variants with mutations at amino acidpositions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332(using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313,325, 327, 328, 329, 330, or 332 (using Kabat numbering), of whichmutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262,263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328,329, 330, or 332 may reduce ADCC activity or reduce binding to an Fcgamma receptor.

Covalent Modifications

Antibodies comprising covalent modifications are also contemplated foruse in the methods. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Other modifications include histidlyl, lysinyl arginyl, tyrosyl,glutaminyl and asparaginyl hydroxylation of proline and lysine. Methodsfor making such modifications are disclosed in U.S. Pat. No. 8,926,976,incorporated herein by reference, and in the art.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R-N.dbd.C.dbd.N-R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 and in Aplin and Wriston, (CRC Crit. Rev.Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal., (Arch. Biochem. Biophys. 259: 52 (1987)) and by Edge et al., (Anal.Biochem. 118: 131 (1981)). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., (Meth. Enzymol. 138:350 (1987)).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art.

Derivatives

As stated above, derivative, when used in connection with antibodysubstances and polypeptides, refers to polypeptides chemically modifiedby such techniques as ubiquitination, conjugation to therapeutic ordiagnostic agents, labeling (e.g., with radionuclides or variousenzymes), covalent polymer attachment such as PEGylation (derivatizationwith polyethylene glycol) and insertion or substitution by chemicalsynthesis of amino acids such as ornithine. Derivatives of theantibodies disclosed herein are also useful as therapeutic agents andmay be used in the methods herein.

The conjugated moiety can be incorporated in or attached to an antibodysubstance either covalently, or through ionic, van der Waals or hydrogenbonds, e.g., incorporation of radioactive nucleotides, or biotinylatednucleotides that are recognized by streptavadin.

Polyethylene glycol (PEG) may be attached to the antibody substances toprovide a longer half-life in vivo. The PEG group may be of anyconvenient molecular weight and may be linear or branched. The averagemolecular weight of the PEG will preferably range from about 2kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEGgroups will generally be attached to the antibody substances of thedisclosure via acylation or reductive alkylation through a natural orengineered reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the antibody substance(e.g., an aldehyde, amino, or ester group). Addition of PEG moieties toantibody substances can be carried out using techniques well-known inthe art. See, e.g., International Publication No. WO 96/11953 and U.S.Pat. No. 4,179,337.

Ligation of the antibody substance with PEG usually takes place inaqueous phase and can be easily monitored by reverse phase analyticalHPLC. The PEGylated substances are purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Antibody Conjugates

An antibody may be administered in its “naked” or unconjugated form, ormay be conjugated directly to other therapeutic or diagnostic agents, ormay be conjugated indirectly to carrier polymers comprising such othertherapeutic or diagnostic agents. In some embodiments the antibody isconjugated to a cytotoxic agent such as a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablechemotherapeutic agents include: daunomycin, doxorubicin, methotrexate,and vindesine (Rowland et al., (1986) supra). Suitable toxins include:bacterial toxins such as diphtheria toxin; plant toxins such as ricin;small molecule toxins such as geldanamycin (Mandler et al., J. Natl.Cancer Inst. 92(19):1573-81 (2000); Mandler et al., Bioorg. Med. Chem.Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13.786-91 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-23 (1996)), auristatins (Doronina et al., Nat.Biotech. 21: 778-84 (2003) and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)).Antibody-Drug Conjugates and methods are reviewed in Ducry L, mAbs.6(1), 2014 and Shen W C, AAPS., 17: 3-7 (2015).

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Sternberger, L. A.et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate. Examples of agents to which the antibody can be conjugatedinclude any of the cytotoxic or chemotherapeutic agents describedherein.

Alternatively, conjugated antibodies can be prepared by directlyconjugating an antibody component with a therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata therapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Antibody Fusion Proteins

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch fusion proteins are ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—ACancer Journal for Clinicians 44:43 (1994). Other suitable toxins areknown to those of skill in the art.

Antibodies of the present disclosure may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to convertit into its more active, cytotoxic form.

Enzymes that are useful in the present disclosure include, but are notlimited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as α-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of thedisclosure into free active drugs (See, e.g., Massey, Nature 328:457-458 (1987). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes above can be covalently bound to the antibodies bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the disclosure linked to at least a functionally activeportion of an enzyme of the disclosure can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312:604-608 (1984)).

Recombinant Production of Antibodies

DNA encoding an antibody described herein may be isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibodies). Usually this requires cloning the DNAor, preferably, mRNA (i.e., cDNA) encoding the antibodies. Cloning andsequencing is carried out using standard techniques, such as for examplepolymerase chain reaction (PCR), (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress; Ausubel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994)), which are incorporated herein by reference).

Sequencing is carried out using standard techniques (see, e.g., Sambrooket al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, ColdSpring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci.USA 74: 5463-5467, which is incorporated herein by reference). Bycomparing the sequence of the cloned nucleic acid with publishedsequences of human immunoglobulin genes and cDNAs, one of skill willreadily be able to determine, depending on the region sequenced, (i) thegermline segment usage of the immunoglobulin polypeptide (including theisotype of the heavy chain) and (ii) the sequence of the heavy and lightchain variable regions, including sequences resulting from N-regionaddition and the process of somatic mutation. One source ofimmunoglobulin gene sequence information is the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, simian COScells, human embryonic kidney 293 cells (e.g., 293E cells), Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

For recombinant production of the antibodies, the nucleic acid encodingit is isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence, which are known and described in the art.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present disclosure, particularlyfor transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

Examples of useful mammalian host cell lines are Chinese hamster ovarycells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies that bind target.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. (Science 240:1041-43, 1988; ICSU ShortReports 10:105 (1990); and Proc. Natl. Acad. Sci. USA 90:457-461 (1993)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. See also, (Carter et al., Bio/Technology10:163-167 (1992).

Alternatively, the antibody can be synthesized in a cell-free systemusing prokaryotic or eukaryotic in vitro translation (see Stech andKubick, Antibodies 4:12-33, 2015).

The antibody composition can be purified using, for example,hydroxylapatite chromatography cation or avian exchange chromatography,and affinity chromatography, with affinity chromatography being thepreferred purification technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Meth. 62: 1-13, 1983). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH 3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE® chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Screening Methods

Effective therapeutics depend on identifying efficacious agents devoidof significant toxicity. Antibodies may be screened for binding affinityby methods known in the art. For example, gel-shift assays, Westernblots, radiolabeled competition assay, co-fractionation bychromatography, co-precipitation, cross linking, ELISA, surface plasmonresonance, KinExA and the like may be used, which are described in, forexample, Current Protocols in Molecular Biology (1999) John Wiley &Sons, NY, which is incorporated herein by reference in its entirety.

Methods for assessing neutralizing biological activity of anti-PTH1Rcompounds are known in the art. See, e.g., International Patentapplication WO 2004073587; Haramoto et al., 2007 Oral Dis. 13(1):23-31.

Additional methods for assessing the biological activity andneutralization of PTH1R (e.g., by PTH1R antibodies) are known in theart. For example, neutralization can be measured by neutralizationassays and expressed as an IC50 value. The IC50 value can be calculatedfor a given molecule by determining the concentration of molecule neededto elicit half inhibition of the maximum biological response of a secondmolecule or cell activity. The lower the IC50, the greater the potencyof the molecule to inhibit the desired protein activity.

Combination Therapy

A PTH1R antibody of the present disclosure may be administered with asecond agent and the combination may be useful to treat a disease ordisorder as described herein. In the case of the use of antibodies toPTH1R, if more than one PTH1R antibody is effective at binding torespective target antigen, it is contemplated that two or moreantibodies to different epitopes of the target antigen may be mixed suchthat the combination of antibodies provides still further improvedefficacy against a condition or disorder to be treated with inhibitorsof PTH1R. Compositions comprising one or more antibody of the inventionmay be administered to persons or mammals suffering from, or predisposedto suffer from, a condition or disorder associated with the targetpolypeptide PTH1R or that will be improved by blocking the activity ofPTH or PTHrP through PTH1R.

Concurrent administration of two therapeutic agents does not requirethat the agents be administered at the same time or by the same route,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

Alternatively, the second agent may be other therapeutic agents, such ascytokines, growth factors, inhibitors and antibodies to other targetantigens.

It is contemplated the therapeutic agents of the present disclosure maybe given simultaneously, in the same formulation. It is furthercontemplated that the agents are administered in a separate formulationand administered concurrently, with concurrently referring to agentsgiven within 30 minutes of each other. It is further contemplated that asecond agent may be given simultaneously.

In another aspect, an antibody to PTH1R is administered prior toadministration of the other composition. Prior administration refers toadministration of an antibody within the range of one week prior totreatment with the other agent, up to 30 minutes before administrationof the other agent. It is further contemplated that an agent isadministered subsequent to administration of another composition oragent. Subsequent administration is meant to describe administrationfrom 30 minutes after antibody treatment up to one week after antibodyadministration. It is further contemplated that a second agent maybeadministered in this manner prior, or subsequent to, administration ofthe PTH1R antibody.

It is further contemplated that other adjunct therapies may beadministered, where appropriate. For example, the patient may also beadministered surgical therapy, chemotherapy, a cytotoxic agent,photodynamic therapy or radiation therapy where appropriate.

It is further contemplated that when the therapeutic agents herein areadministered in combination with a second agent, such as for example,wherein the second agent is a cytokine or growth factor, or achemotherapeutic agent, the administration also includes use of aradiotherapeutic agent or radiation therapy. The radiation therapyadministered in combination with an antibody composition is administeredas determined by the treating physician, and at doses typically given topatients being treated for cancer.

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., I131, I125, Y90 andRe186), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the disclosure.

In one embodiment the second agent is denosumab (Xgeva®, Prolia®). Inother embodiments the second agent is a bisphosphonate (e.g. zoledronicacid). In other embodiments the second agent is a calcitonin.

Chemotherapeutic and other agents contemplated for use with theantibodies of the present disclosure include, but are not limited tothose listed in Table 1:

TABLE 1 Chemotherapeutic and other agents contemplated for use with theantibodies of the present disclosure Alkylating agents Natural productsNitrogen mustards Antimitotic drugs mechlorethamine Taxanescyclophosphamide paclitaxel ifosfamide Vinca alkaloids melphalanvinblastine (VLB) chlorambucil vincristine Nitrosoureas vinorelbinecarmustine (BCNU) Taxotere ® (docetaxel) lomustine (CCNU) estramustinesemustine (methyl-CCNU) estramustine phosphateEthylenimine/Methyl-melamine Epipodophylotoxins thriethylenemelamine(TEM) etoposide triethylene thiophosphoramide teniposide (thiotepa)Antibiotics hexamethylmelamine actimomycin D (HMM, altretamine)daunomycin (rubido-mycin) Alkyl sulfonates doxorubicin (adria-mycin)busulfan mitoxantroneidarubicin Triazines bleomycin dacarbazine (DTIC)splicamycin (mithramycin) Antimetabolites mitomycinC Folic Acid analogsdactinomycin methotrexate aphidicolin Trimetrexate Enzymes PemetrexedL-asparaginase (Multi-targeted antifolate) L-arginase Pyrimidine analogsRadiosensitizers 5-fluorouracil metronidazole fluorodeoxyuridinemisonidazole gemcitabine desmethylmisonidazole cytosine arabinosidepimonidazole (AraC, cytarabine) etanidazole 5-azacytidine nimorazole2,2′-difluorodeoxy-cytidine RSU 1069 Purine analogs EO9 6-mercaptopurineRB 6145 6-thioguanine SR4233 azathioprine nicotinamide2′-deoxycoformycin 5-bromodeozyuridine (pentostatin) 5-iododeoxyuridineerythrohydroxynonyl-adenine bromodeoxycytidine (EHNA) Miscellaneousagents fludarabine phosphate bisphosphonates 2-chlorodeoxyadenosineRANKL inhibitor (cladribine, 2-CdA) denosumab Type I TopoisomeraseInhibitors Platinium coordination camptothecin complexes topotecancisplatin irinotecan carboplatin Biological response modifiersoxaliplatin G-CSF nthracenedione GM-CSF mitoxantrone DifferentiationAgents Substituted urea retinoic acid derivatives hydroxyurea Hormonesand antagonists Methylhydrazine derivativesAdrenocorticosteroids/antagonists N-methylhydrazine (MIH) calcitoninprocarbazine prednisone and equiv-alents Adrenocortical suppressantdexamethasone mitotane (o,p′- DDD) ainoglutethimide ainoglutethimideProgestins Cytokines hydroxyprogesterone caproate interferon (α, β, γ)medroxyprogesterone acetate interleukin-2 megestrol acetatePhotosensitizers Estrogens hematoporphyrin derivativesdiethylstilbestrol Photofrin ® ethynyl estradiol/equivalentsbenzoporphyrin derivatives Antiestrogen Npe6 tamoxifen tin etioporphyrin(SnET2) Androgens pheoboride-a testosterone propionatebacteriochlorophyll-a fluoxymesterone/equivalents naphthalocyaninesAntiandrogens phthalocyanines flutamide zinc phthalocyaninesgonadotropin-releasing Radiation hormone analogs X-ray leuprolideultraviolet light Nonsteroidal antiandrogens gamma radiation flutamidevisible light infrared radiation microwave radiation

Treatment of Disorders

In another embodiment, any of the types of antibodies described hereinmay be used in the methods. In exemplary embodiments, the targetspecific antibody is a human, chimeric or humanized antibody. In anotherexemplary embodiment, the target is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses atarget protein that target specific antibody cross-reacts with. Theantibody may be administered to a non-human mammal expressing a targetprotein with which the antibody cross-reacts (e.g. a primate) forveterinary purposes or as an animal model of human disease.

In one embodiment, the disclosure provides a method for treatinghypercalcemia associated with increased PTH or PTHrP protein expressioncomprising the step of administering to a subject in need thereof atherapeutically effective amount of a PTH1R antibody or a pharmaceuticalcomposition contemplated herein.

In another embodiment, the disclosure provides a method for treating adisease, condition or disorder associated with increased parathyroidhormone expression, increased parathyroid hormone related proteinexpression or increased PTH1R expression comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or a pharmaceutical composition contemplatedherein.

In another embodiment, the disclosure provides a method for treating adisease, condition or disorder selected from the group consisting ofcancer, PTH- or PTHrP-induced hypercalcemia, Humoral Hypercalcemia ofMalignancy (HHM), familial hypocalciuric hypercalcemia, tuberculosis,sarcoidosis, Primary Hyperparathyroidism (PHPT), SecondaryHyperparathyroidism (SHPT) and cachexia.

In various embodiments, administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein ameliorates one or more symptoms ofhypercalcemia.

In various embodiments, administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein ameliorates one or more symptoms includewasting syndrome in PTHrP induced HHM, extension of HHM survival due toreduced hypercalcemia and/or wasting syndrome. PTHrP and PTH1R have beenimplicated in breast cancer and gastric cancer progression (Hoey et al.,2003 Br J Cancer. 88(4): 567-573; Ito et al., 1997 J Gastroenterol.32(3):396-400). Furthermore overexpression of PTHrP and PTH1R in breasttumor cells has been shown to promote the growth of such cells inskeletal metastases by stimulating their proliferation in an autocrinefashion (Hoey et al., 2003 Br J Cancer. 88(4): 567-573).

In one embodiment, the disclosure provides a method for treating canceror preventing the recurrence of cancer comprising administering to asubject in need thereof a therapeutically effective amount of a PTH1Rantibody or a pharmaceutical composition as contemplated herein.

Exemplary conditions or disorders that can be treated with antibodies ofthe present disclosure include cancers, such as a cancer selected fromthe group consisting of bone cancer, lung cancer, hepatocellular cancer,pancreatic cancer, kidney cancer, fibrotic cancer, breast cancer,myeloma, squamous cell carcinoma, colorectal cancer and prostate cancer.In related aspects the cancer is metastatic. In a related aspect, themetastasis includes metastasis to the bone or skeletal tissues, liver,lung, kidney or pancreas. It is contemplated that the methods hereinreduce tumor size or tumor burden in the subject, and/or reducemetastasis in the subject. In various embodiments, the methods reducethe tumor size by 10%, 20%, 30% or more. In various embodiments, themethods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

In one embodiment, treatment of cancer in an animal in need of saidtreatment, comprises administering to the animal an effective amount ofan antibody of PTH1R or a composition comprising an antibody describedherein.

The conditions treatable by methods of the present disclosure preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Formulation of Pharmaceutical Compositions

To administer antibodies of the present disclosure to human or testanimals, it is preferable to formulate the antibodies in a compositioncomprising one or more pharmaceutically acceptable carriers. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce allergic, or other adversereactions when administered using routes well-known in the art, asdescribed below. “Pharmaceutically acceptable carriers” include any andall clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the antibodies may be suitably administeredby pulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous, intra muscular or subcutaneous injections, depending inpart on whether the administration is brief or chronic. Otheradministration methods are contemplated, including topical, particularlytransdermal, transmucosal, rectal, oral or local administration e.g.through a catheter placed close to the desired site.

Pharmaceutical compositions of the present disclosure containing theantibodies described herein as an active ingredient may containpharmaceutically acceptable carriers or additives depending on the routeof administration. Examples of such carriers or additives include water,a pharmaceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer,carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptablesurfactant and the like. Additives used are chosen from, but not limitedto, the above or combinations thereof, as appropriate, depending on thedosage form of the present disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the antibody to be administered canbe prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers.

A variety of aqueous carriers, e.g., sterile phosphate buffered salinesolutions, bacteriostatic water, water, buffered water, 0.4% saline,0.3% glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Therapeutic formulations of the antibodies are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Aqueous suspensions may contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

The antibodies described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

The PTH1R antibodies described herein can be prepared and administeredas a co-formulation with one or more additional antibodies. In oneaspect, at least two of the antibodies recognize and bind differentantigens. In another aspect, at least two of the plurality of antibodiescan specifically recognize and bind different epitopes of the sameantigen.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of antibody in these formulations can vary widely, forexample from less than about 0.5%, usually at or at least about 1% to asmuch as 15 or 20% by weight and will be selected primarily based onfluid volumes, viscosities, etc., in accordance with the particular modeof administration selected. Thus, a typical pharmaceutical compositionfor parenteral injection could be made up to contain 1 ml sterilebuffered water, and 50 mg of antibody. A typical composition forintravenous infusion could be made up to contain 250 ml of sterileRinger's solution, and 150 mg of antibody. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed., Mack PublishingCompany, Easton, Pa. (1980). An effective dosage of antibody is withinthe range of 0.01 mg to 1000 mg per kg of body weight peradministration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

Antibody compositions contemplated for use to inhibit target activity,including binding of the target to its cognate receptor or ligand,target-mediated signaling, and the like. In particular, the compositionsexhibit inhibitory properties at concentrations that are substantiallyfree of side effects, and are therefore useful for extended treatmentprotocols. For example, co-administration of an antibody compositionwith another, more toxic, cytotoxic agent can achieve beneficialinhibition of a condition or disorder being treated, while effectivelyreducing the toxic side effects in the patient.

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the present disclosure are wellbalanced, thereby enhancing their utility for both in vitro andespecially in vivo uses, while other compositions lacking such balanceare of substantially less utility. Specifically, compositionscontemplated for use in the disclosure have an appropriate degree ofsolubility in aqueous media which permits absorption and bioavailabilityin the body, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membrane to a putative siteof action. Thus, antibody compositions contemplated are maximallyeffective when they can be delivered to the site of target antigenactivity.

Administration and Dosing

In one aspect, methods of the present disclosure include a step ofadministering a pharmaceutical composition. In certain embodiments, thepharmaceutical composition is a sterile composition.

Methods of the present disclosure are performed using anymedically-accepted means for introducing therapeutics directly orindirectly into a mammalian subject, including but not limited toinjections, oral ingestion, intranasal, topical, transdermal,parenteral, inhalation spray, vaginal, or rectal administration. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intracisternal injections, as well as catheter orinfusion techniques. Administration by, intradermal, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection andor surgical implantation at a particular site is contemplated as well.

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the disclosure implanted near or at site of the cancer.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, once per week, once every 2 weeks, twice per month, oncemonthly, once every two months, or once every three months, or at alonger interval.

The amounts of antibody composition in a given dosage may vary accordingto the size of the individual to whom the therapy is being administeredas well as the characteristics of the disorder being treated. Inexemplary treatments, it may be necessary to administer about 1 mg/day,5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000 mg/day. Theseconcentrations may be administered as a single dosage form or asmultiple doses. Standard dose-response studies, first in animal modelsand then in clinical testing, reveals optimal dosages for particulardisease states and patient populations.

Also contemplated in the present disclosure, the amounts of PTH1Rantibody in a given dosage may vary according to the size of theindividual to whom the therapy is being administered as well as thecharacteristics of the disorder being treated. The antibody compositionscan be administered in a dose range of 0.1 to 15 mg, twice weekly as anintravenous infusion over 30-60 minutes every 1, 2 or 4 weeks untildisease progression or unacceptable toxicity. In various embodiments,the antibody compositions can be administered intravenously,subcutaneously or intramuscularly, in a dose range of 0.3-30 mg/kg twiceweekly or every 1, 2 or 4 weeks. In various embodiments, the dose can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 30 mg/kg. In various embodiments, theantibody compositions can be administered intravenously in a dose rangeof 0.3-3 mg/kg, 1 to 6 mg/kg or 2 to 6 mg/kg twice weekly or every 1, 2or 4 weeks. Alternatively, the antibody compositions can be administeredintravenously, subcutaneously or intramuscularly in a dose range of0.5-5 mg/kg twice weekly or every 1, 2 or 4 weeks.

It will also be apparent that dosing may be modified if additionaltherapeutics are administered in combination with therapeutics of thedisclosure.

Kits

As an additional aspect, the disclosure includes kits which comprise oneor more compounds or compositions packaged in a manner which facilitatestheir use to practice methods of the disclosure. In one embodiment, sucha kit includes a compound or composition described herein, packaged in acontainer such as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of the compoundor composition in practicing the method. Preferably, the compound orcomposition is packaged in a unit dosage form. The kit may furtherinclude a device suitable for administering the composition according toa specific route of administration or for practicing a screening assay.Preferably, the kit contains a label that describes use of the antibodycomposition.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Example 1: Discovery of Antibodies that Bind to PTH1R

A. Phage Panning and Rescue

In order to identify antibodies that bind the PTH1R, three rounds ofcell panning using naïve scFv or Fab antibody phage libraries (Schwimmeret al. 2013 J. Immun. Meth. 391(1-2):60-71) were performed. Kappa andlambda chain libraries were panned together. For the first round ofphage panning, about 50× library equivalents were blocked at RT for 1 hrin 5% FCS/PBS in the presence of the FLAG peptide (Sigma Aldrich).Non-specific binders to CHO-K1 (parental cells) were then deselectedagainst 5×10⁷ CHO-K1 cells that did not express the PTH1R receptor.Subsequently, deselected cells were allowed to bind to 1.5×10⁸ CHO-PTH1Rcells expressing the full-length human receptor in the presence of theFLAG peptide for 2 hr at 37° C. The receptor-bound phage were thenwashed with 5% FCS-PBS and then with PBS. The duration of washes wasincreased in each successive round of panning in order to elevate thestringency of selection. Bound phage was then eluted via the addition of100 mM Trimethylamine (TEA) elution buffer (Merck-TX1200-5) andneutralized with an equal volume of 1 M Tris-HCl, pH 7.4. Eluted andneutralized phage was then used to infect TG1 bacterial cells expressingthe cytFkpA chaperone variant (Levy et al. 2013 J. Immun. Meth.394(1-2):10-21) at OD600 ˜0.5. Infection was performed at 37° C. for 1hr with shaking at 100 rpm. Cells were then plated on 2YT agarose platesthat were supplemented with 100 μg/mL carbenicillin, 34 μg/mlchloramphenicol and 2% glucose and incubated overnight at 30° C.

In order to rescue phage for the next round of panning, 50×-100× outputwas superinfected with the helper phage M13K07 (New England Biolabs,MA). Cells were harvested and allowed to grow at 37° C., starting fromOD600-0.05. When the OD600 nm ˜0.5, cells were infected with M13K07 atmultiplicity of infection (MOI) ˜20, at 37° C. for 1 hour with rotationat 100 rpm. Following infection, cells were pelleted and transferred tonew 2YT media supplemented with 50 μg/mL kanamycin, 100 μg/mLcarbenicillin and 0.2% arabinose in order to allow the expression of thechaperone cytFkpA. Following overnight growth of bacterial cultures at25° C., phage was harvested following centrifugation at 4° C. and thenPEG-precipitated to be used as input for the next round of panning.Selection enrichment was monitored by the amount of phage input used foreach panning round versus the resulting phage output titer.

Alternatively, rescued phage resulting from the second round of panning,were allowed to bind to CHO cells engineered to express human PTH1R.Phage bound to CHO-PTH1R cells were then isolated using cell sortingmethods based on those described by Siegel et al., 2002 Methods Mol.Biol. 178: 219-226.

B. Screening

Library panning outputs were screened by FACS. Plates containingperiplasmic bacterial extracts of selected clones were prepared usingstandard protocols. They were then screened by FACS for binding to CHOcells expressing human PTH1R with a FLAG tag on the N-terminus, versusbinding to parental CHO-K1 cells. An anti-FLAG tag monoclonal antibodywas used as a positive control to detect PTH1R-expressing cells.

Antibody fragments binding PTH1R were then reformatted into human IgG1,IgG2 or IgG4 antibodies. The variable heavy (VH) and light (VL) chainsof the selected scFv or Fab fragments were amplified by PCR, cloned intoplasmid vectors containing the antibody constant region sequences, andtransiently transfected into 293E cells using standard methods togenerate IgG2 antibodies for further characterization. Reformattedantibodies were tested for PTH1R binding and in functional assays, asdescribed below.

Example 2: Affinity Maturation

Based on initial binding and functional characterization studies, twoantibodies, XPA.85.012 and XPA.85.017 were selected for affinitymaturation using light chain shuffling.

A. Selection of the XPA.85.012 Affinity-Matured Variants Light ChainLibrary Construction

The VH region of XPA.85.012 was cloned into plasmid DNA containing twodifferent kappa chain Fab libraries. The resulting library sizes for theresulting libraries, XPA85.012.010 and XPA85.012.050, were 7.6×10⁸ cfuand 2.1×10⁹ cfu respectively. Each library was rescued (50x libraryequivalents) using TG1+cytFkpA cells (Levy et al. 2013 J. Immun. Meth.394(1-2):10-21).

Phage Panning and Rescue

Recombinant N-terminal extracellular domain of the human PTH1R receptor(N-ECD PTH1R, R&D Systems, MN) was biotinylated with EZ-LinkNHS-PEG4-Biotin (Thermo Scientific, Rockford, Ill.) using themanufacturer's protocol and 20-fold molar excess of biotin reagent.Non-reactive NHS-PEG4-Biotin was removed by spinning twice with Zeba™Spin Desalting columns (7 K MWCO) (Thermo Scientific, Rockford, Ill.).The biotinylation of N-ECD PTH1R was confirmed by surface plasmonresonance (SPR).

For the first round of phage panning, 100× library equivalents ofXPA85.012.010 and XPA85.012.050 (7.6×10¹⁰ cfu and 2.1×10¹¹ cfu,respectively) were blocked on ice for 1 hr in 1 mL of 3% BSA/PBS.Binders to streptavidin were deselected from blocked phage by addingblocked phage to streptavidin-coated magnetic beads (Dynabeads™ M-280Streptavidin, Thermo Fisher Scientific, Carlsbad, Calif.) and incubatedwith rotation for 45 minutes at room temperature. The deselection stepwas repeated once more. A magnet was used to separate beads from phage.Concurrent to the deselection steps, 40 pmoles of biotinylated N-ECDPTH1R was allowed to bind to streptavidin-coated magnetic beads byincubating at room temperature with rotation for 45 minutes. Selectionwas performed by adding deselected phage to biotinylated N-ECD PTH1Rbound to magnetic streptavidin beads and incubated with rotation for 1.5hours. After selection, unbound phage was removed by three 5 minutewashes with 3% BSA/PBS+0.1% Tween-20, followed by three 5 minute washeswith 3% BSA/PBS and one quick wash with PBS. Bound phage was eluted frombeads after the final wash step by the addition of 100 mM TEA withrotation at room temperature for 20 minutes. Eluted phage wasneutralized with the addition of equal volume 1 M Tris-HCl, pH 7.4 andbeads were neutralized with the addition of 1 mL 1 M Tris-HCl, pH 7.4.Eluted and bead-bound neutralized phage were used separately to infectlog growing TG1+cytFkpA bacterial cells (OD600 ˜0.5). Infection was at37° C. for 30 min without shaking, followed by 30 min additionalincubation at 37° C. with shaking at 100 rpm. Cells were plated on 2YTmedia supplemented with 100 μg/mL carbenicillin, 34 μg/mLchloroamphenicol and 2% glucose (2YTCCG) agar bioassay plates andincubated overnight at 30° C. to allow for overnight lawn growth. Allplates were scraped and cells were combined to make bacterial glycerolstocks.

In preparation for use as input for the next round, 100× of previousround output was rescued by superinfection using M13K07 helper phage(New England Biolabs, Carlsbad, Calif.). This was done by inoculating2YTCCG media with cells scraped from previous panning round output.OD600 nm was measured for starting culture and adjusted to reflect astarting OD600 nm of ˜0.05. Cells were grown at 37° C. with shakinguntil cells reached log-growing phase of OD600 nm ˜0.5. Cells wereinfected with M13K07 helper phage at a multiplicity of infection(MOI)=˜20, at 37° C. for 30 min without shaking, followed by anadditional 30 min incubation at 37° C. with shaking at 100 rpm. Afterinfection at 37° C., cells were pelleted and transferred to new 2YTmedia supplemented with 25 μg/mL kanamycin, 100 μg/mL carbenicillin and0.2% L-arabinose. Cultures were grown overnight at 25° C. Phage wasseparated from cells and debris by centrifugation and resultingsupernatant was recovered and PEG-6000/NaCl precipitated. Selectionenrichment was monitored by the amount of input used for each panninground and the resulting phage output titer.

For the second and third panning rounds, the same solution phaseprotocols followed in round one were used with the following exceptions.Phage input amount used in panning round two were ˜1.0×10¹⁰ cfu and forround three ˜4.0×10⁸ to 5×10¹⁰ cfu. For round two, 10 pmoles ofbiotinylated antigen was used in selection, and for round three, 1.0 and0.4 pmoles of biotinylated antigen was used. In round two, the washconditions were five 5 minute washes with 3% BSA/PBS+0.1% Tween-20,followed by five 5 minute washes with 3% BSA/PBS and one quick wash withPBS. In round three, two wash conditions were implemented using theKingfisher instrument (Thermo Fisher Scientific) to wash unbound phagefrom beads after selections. In the first wash condition, the Kingfisherwas programmed to wash beads 8 times with 3% BSA/PBS-0.1% Tween-20 for 4minutes followed 8 times with 3% BSA/PBS for 4 minutes ending with a 1ml PBS wash. Bound phage were collected using the magnet and eluted aspreviously mentioned with eluted and bead bound phage kept separate forTG1+cytFpKA infections, bacterial glycerol stocks and the picking ofsingle clones. In the second wash condition, the Kingfisher wasprogrammed to wash beads 5 times with 3% BSA/PBS-0.1% Tween-20 for 4minutes followed 5 times with 3% BSA/PBS for 4 minutes ending with a 1ml 1% BSA/PBS wash at 4° C. with rotation overnight followed by onequick PBS wash. Bound phage were collected using the magnet and elutedas previously mentioned with eluted and bead bound phage kept separatefor TG1+cytFkpA infections, bacterial glycerol stocks and the picking ofsingle clones.

B. Selection of the XPA.85.017 Affinity-Matured Variants Light ChainLibrary Construction

The VH region of XPA.85.017 was cloned into plasmid DNA containing twodifferent lambda chain Fab libraries. The resulting library sizes forthe resulting libraries, XPA.85.017.010 and XPA.85.017.031, were8.21×10⁸ cfu and 7.51×10⁸ cfu respectively. Each library was rescued(50× library equivalents) using TG1+cytFkpA (Levy et al., 2013 J. Immun.Meth. 394(1-2):10-21)

Phage Panning and Rescue

Panning was performed on recombinant N-terminal extracellular domain ofhuman PTH1R as described above except that for the first round of phagepanning, concurrent to the deselection steps, 10 pmoles rather than 40pmoles of biotinylated N-ECD PTH1R was allowed to bind tostreptavidin-coated magnetic beads.

For the second and third panning rounds, the same solution phaseprotocols followed in round one above were used with the followingexceptions. Phage input amount used in panning rounds two and three was˜1.0×10¹⁰ cfu. For round two, 1 pmoles of biotinylated antigen was usedin selection; for round three, 0.5 and 0.1 pmoles of biotinylatedantigen was used. An additional panning condition was introduced inround 3 using a 30 min selection (rather than 1.5 hr) and 0.5 pmoles.Following the second and third rounds of selection, the Kingfisherinstrument was used to wash unbound phage from beads. In round two, theKingfisher was programmed to wash beads 5 times with PBS-0.1% TWEEN+3%BSA for 5 minutes followed by 5 times with 3% BSA/PBS for 5 minutes. Inround three panning, beads were washed 7 times with PBS-0.1% TWEEN+3%BSA for 5 minutes, followed by 7 times with 1×PBS+3% BSA 5 minute washesand a final quick wash with PBS. An overnight wash with 500× fold excesscold N-ECD PTH1R antigen was added to the 0.5 and 0.1 pmoles arms withvarying duration of selection.

C. Screening

Bacterial periplasmic extracts containing secreted antibody fragmentsfor use in screening for PTH1R binders were prepared by standardmethods. In order to assess the improved binding kinetics of the lightchain shuffled mutants, off-rate screening of crude antibody fragmentswas performed using surface plasmon resonance (SPR). The primaryantibody panning and antibody discovery had utilized CHO-K1 cellsover-expressing the full length human PTH1R. Further testingdemonstrated that both the XPA.85.012 and XPA.85.017 clones bound to asoluble recombinant N-Terminal extracellular domain (ECD) which could beutilized in SPR based high throughput screening and affinitycharacterization.

The SPR screening assays were performed on a BIACORE 4000 (GEHealthcare) using a direct binding assay format. In these assays, a CMSBIACORE chip was prepared via standard amine coupling chemistry usingthe BIACORE Amine Coupling kit (GE Healthcare, Piscataway, N.J.). Thehuman PTH1R N-terminal ECD (R&D Systems) was diluted to 4 μg/mL inacetate pH 4.5 and injected for 5 minutes. This immobilizesapproximately 1500 RU of ECD. Periplasmic extracts were diluted 1:1 withHBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v SurfactantP20) (Teknova, Hollister Calif.) with 5 mg/mL BSA and filtered through a0.2 μM Millex GV filter plate (Millipore) and then injected at 30uL/minute for 240 seconds with a 300 second dissociation. Regenerationafter each PPE injection was 20 seconds of 10 mM Glycine pH 3.0. Therunning buffer used was HBS-EP+ with 2.5 mg/mL BSA (Sigma Aldrich, St.Louis Mo.). The stability early report point in the BIACORE 4000software was used to evaluate PPE binding levels and dissociation (kd)was determined.

Selected clones with improved off-rates were reformatted as IgG1, IgG2or IgG4 antibodies as described in Example 1. FIGS. 1A and 1B showmultiple sequence alignments (MSA) of the light chain amino acids offamilies for children of 12 (FIG. 1A) or families for children of 17(FIG. 1B), were performed against the parent sequence using the ClustalWalgorithm.

Example 3: IgG Affinity Measurement by SPR

In order to measure the on-rate (k_(a)) and off-rate (k_(d)) constantsfor the various reformatted antibodies an SPR approach was pursued.

An anti-human IgG-Fc CM5 sensor chip (GE Healthcare) was prepared usingstandard amine coupling as described by the manufacturer's protocol forthe capture Ab on a BIACORE 4000 system (GE Healthcare). Briefly, thechip surface was activated with a 10 minute injection at 10 μL/minute ofa freshly mixed 1:1 solution of 0.1 M N-Hydroxysuccinimide (NHS) and 0.4M 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).Following the activation injection, 15 ug/mL anti-human IgG-Fc antibodyin acetate pH 4.5 was injected at 10 μL/minute for 10 minutes to achievea high density capture surface. 7 minutes of 1M Ethanolaminehydrochloride-NaOH pH 8.5 was injected to block the surface. The NHS,EDC, and Ethanolamine used were from the BIACORE Amine Coupling Kit.

Kinetic Analysis was performed using HBS-EP+ (Teknova) with 2.5 mg/mLBSA (Sigma Aldrich, St. Louis Mo.). Antibodies were diluted to 0.5 μg/mLand captured to achieve a surface density of 100 to 200 RU. PTH1RN-terminal ECD samples were injected at four concentrations of 200, 50,12.5, and 3.125 nM as well as blank injections. Injections wereperformed at 30 μL/minute for 240 seconds with a 360 second dissociationtime. Regeneration was then performed with one 60 second injection 3MMgCl.

The data were analyzed using BIACORE 4000 Evaluation Software and wasdouble referenced by subtracting both the blank flow cell data and theaveraged bracketing blank injections. The data was fit by simultaneouslyfitting the an off-rate (k_(d)) and on-rate (k_(a)), and calculating theK_(D) as K_(D)=k_(d)/k_(a). Measured rate constants are shown in Table 2and representative data is included in FIG. 2.

TABLE 2 SPR Based Affinity Estimates Clone k_(a) (1/Ms) k_(d) (1/s)K_(D) (M) XPA.85.012 1.3E+06 2.5E−02 2.0E−08 XPA.85.017 3.5E+05 8.6E−042.5E−09 XPA.85.287 3.6E+06 3.6E−04 9.9E−11 XPA.85.288 7.8E+05 1.4E−041.8E−10 XPA.85.326 3.8E+05 7.3E−05 1.9E−10 XPA.85.327 7.4E+05 1.1E−041.5E−10 XPA.85.328 5.6E+05 1.8E−04 3.2E−10 XPA.85.329 3.4E+05 7.6E−052.2E−10 XPA.85.330 4.7E+05 9.0E−05 1.9E−10 XPA.85.331 1.6E+06 3.5E−042.2E−10 XPA.85.332 2.4E+06 1.7E−03 7.1E−10 XPA.85.333 1.8E+06 5.4E−043.0E−10 XPA.85.334 8.6E+05 2.1E−04 2.5E−10 XPA.85.335 8.0E+05 1.9E−032.4E−09 XPA.85.336 1.1E+06 1.9E−02 1.7E−08 XPA.85.337 6.1E+05 4.6E−047.5E−10 XPA.85.338 1.3E+06 5.7E−04 4.3E−10 XPA.85.339 2.9E+06 6.5E−042.2E−10 XPA.85.340 1.4E+06 1.1E−03 7.5E−10 XPA.85.341 1.9E+06 6.0E−043.1E−10 XPA.85.342 2.3E+06 1.2E−03 5.2E−10 XPA.85.343 1.1E+06 4.5E−044.2E−10 XPA.85.344 1.3E+06 2.3E−04 1.8E−10 XPA.85.345 2.3E+06 1.3E−035.6E−10 XPA.85.346 4.5E+05 1.4E−04 3.1E−10 XPA.85.347 8.8E+05 7.7E−048.7E−10

The affinity data as measured in this assay using captured antibodiesdemonstrated increased affinity binding (lower K_(D)) of the affinitymatured variants of both the XPA.85.012 and the XPA.85.017 clones to thehuman recombinant ECD.

Example 4: FACS Binding Assays

A. Binding of XPA.85.012 and XPA.85.017 on CHOK1 and CHO Human PTH1RCells

To assess antibody binding specificity of XPA.85.012 and XPA.85.017 tohuman PTH1R, CHO cells overexpressing human PTH1R were used in a flowbased assay. Binding data for XPA.85.012 and XPA.85.017 is shown FIGS.3A and 3B, respectively. CHOK1 parent cells were also used as a negativecell line. Cells were plated in a 96-well v-bottom plate (Costar: FisherScientific, Waltham, Mass.) with increased concentrations of antibodies.Cells and antibodies were incubated for 30 minutes at 4° C. Followingtwo washes with FACS Buffer (0.5% BSA+0.1% NaN3 in DPBS), cells wereincubated with R-Phycoerythrin AffiniPure F(ab′)₂ Fragment GoatAnti-Human IgG (H+L) (Jackson ImmunoResearch, West Grove, Pa.) for 20minutes at 4° C. Cells were washed twice and resuspended in FACS Buffer.Samples were acquired using BD Cytometer (BD Biosciences, San Jose,Calif.) and data were analyzed using FlowJo (FlowJo, LLC, Ashland,Oreg.) and Prism (GraphPad Software, La Jolla, Calif.).

B. XPA.85.012 and XPA.85.017 Binding Affinity in the Presence andAbsence of PTH and PTHrP

To assess antibody differential binding to the PTH1R ligands, increasingconcentrations of anti-PTH1R antibodies were incubated with CHO humanPTH1R cells for 60 minutes in the presence or absence of saturatingconcentration of PTH and PTHrP. Binding data for XPA.85.012 andXPA.85.017 is shown FIGS. 4A and 4B, respectively. Following two washeswith FACS Buffer (0.5% BSA+0.1% NaN3 in DPBS), cells were incubated withR-Phycoerythrin AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG (H+L)(Jackson ImmunoResearch, West Grove, Pa.) for 20 minutes at 4° C. Cellswere then washed twice and resuspended in FACS Buffer. Samples wereacquired using BD Cytometer (BD Biosciences, San Jose, Calif.) and datawere analyzed using FlowJo (FlowJo, LLC, Ashland, Oreg.) and Prism(GraphPad Software, La Jolla, Calif.).

C. Kinetics (On Rate) of XPA.85.012 and XPA.85.017

SPR data showed that XPA.85.012 has a faster on rate than XPA.85.017. Aflow based assay was developed to confirm the on rate of bothantibodies. First, XPA.85.017 on rate was assessed by flow cytometry(FIG. 5A). Increasing concentrations of XPA.85.017 were incubated withCHO human PTH1R cells for a certain amount of time, from 5 minutes to 24hours. Antibody binding for XPA.85.012 is shown in FIG. 5B. In summary,XPA.85.012 can bind within 1 hr whereas it can take XPA.85.017 24 hr tobind. All time points were stopped at the same time except for the 24 hrsamples. Cells were then washed twice with FACS Buffer (0.5% BSA+0.1%NaN3 in DPBS) and incubated with Allophycocyanin (APC) AffiniPureF(ab′)₂ Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch, WestGrove, Pa.) for 20 minutes at 4° C. Following two washes, cells wereresuspended in FACS Buffer. Samples were acquired using BD Cytometer (BDBiosciences, San Jose, Calif.) and data were analyzed using FlowJo(FlowJo, LLC, Ashland, Oreg.) and Prism (GraphPad Software, La Jolla,Calif.).

D. Assessment of Species Cross Reactivity of Anti-PTH1R Antibodies

CHOK1 overexpressing PTH1R orthologs and endogenous cell lines were usedto assess species cross-reactivity. CHO human PTH1R and CHO mouse PTH1Rcells were generated in house and UMR106 and Saos-2 were purchased fromATCC. UMR106 and Saos-2 are both osteosarcoma cell line expressing therat and human PTH1R, respectively. All four cell lines were used toassess species cross reactivity.

Anti-PTH1R antibodies were screened for both binding to the cell linesexpressing PTH1R orthologs and for differential binding in the presenceor absence of the ligands (PTH or PTHrP). To assess antibody binding anddifferential binding, increasing concentrations of anti-PTH1R antibodieswere incubated with all four cell lines in the presence or absence ofsaturating concentration of PTH and PTHrP. Binding data is shown forXPA.85.017 and XPA.85.012 antibodies to CHO cells expressing human PTHRin the presence and absence of PTH (FIG. 6A) or PTHrP (FIG. 6B). Bindingdata is shown for XPA.85.017 and XPA.85.012 antibodies to CHO cellsexpressing mouse PTHR in the presence and absence of PTH (FIG. 6C) orPTHrP (FIG. 6D).

Antibodies and cells were incubated for 24 hours to allow the sloweron-rate antibody, XPA.85.017, to reach equilibrium. Following two washeswith FACS Buffer (0.5% BSA+0.1% NaN3 in DPBS), cells were incubated withPhycoerythrin AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG (H+L)(Jackson ImmunoResearch, West Grove, Pa.) for 20 minutes at 4° C. Cellswere then washed twice and resuspended in FACS Buffer. Samples wereacquired using BD Cytometer (BD Biosciences, San Jose, Calif.) and datawere analyzed using FlowJo (FlowJo, LLC, Ashland, Oreg.) and Prism(GraphPad Software, La Jolla, Calif.).

E. Binding of Affinity Matured XPA.85.017 Variants to CHOK1, CHO HumanPTH1R, and CHO Mouse PTH1R at Equilibrium Condition in the Presence orAbsence of PTH1R Ligands

To characterize antibody binding to PTH1R, cells were labeled witheither CellTrace™ CFSE or CellTrace™ Violet. Unlabeled CHOK1 parent, CHOhuman PTH1R labeled with CellTrace™CFSE, and CHO mouse PTH1R labeledwith CellTrace™Violet were pooled together and stained with increasingconcentrations of antibody. Antibodies were incubated for 24 hr at 4° C.followed by two washes with FACS Buffer (0.5% BSA, 0.1% NaN3 in DPBS).Cells were then incubated with Allophycocyanin (APC) AffiniPure F(ab′)₂Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch, West Grove,Pa.) for 20 minutes at 4° C. Followed by two washes, cells wereresuspended in FACS Buffer. Samples were acquired using BD Cytometer (BDBiosciences, San Jose, Calif.) and data were analyzed using FlowJo(FlowJo, LLC, Ashland, Oreg.) and Prism (GraphPad Software, La Jolla,Calif.). EC50 Values of antibodies binding to CHO human PTH1R and CHOmouse PTH1R cells are shown in Table 3. Selected XPA.85.017 antibodyvariants were assessed for differential binding on CHO human and CHOmouse PTH1R cells and showed a decrease in binding when in the presenceof either of the PTH1R ligands. Lower EC50 values were observed in theabsence of the ligands (Table 4).

TABLE 3 XPA.85.017 Variants EC₅₀ Values on CHO Human PTH1R and CHO MousePTH1R cells at equilibrium condition. EC₅₀ [nM] anti-PTHR Ab CHO HumanPTHR CHO Mouse PTHR XPA.85.012 0.7926 0.8866 XPA.85.017 0.7183 1.047XPA.85.287 0.04398 0.2163 XPA.85.335 0.2651 0.384 XPA.85.336 0.341614.71 XPA.85.337 1.265 3.273 XPA.85.338 0.1806 0.6804 XPA.85.339 0.14230.2338 XPA.85.340 0.1674 0.322 XPA.85.341 0.202 0.3425

TABLE 4 Differential Binding Profiles for Selected XPA.85.017 Variants.EC₅₀ [nM] CHO Human PTHR CHO Mouse PTHR Antibody Ab + 1 Ab + 3 AntibodyAb + 1 Ab + 3 anti-PTHR Ab Alone uM PTH uM PTHrP Alone uM PTH uM PTHrPXPA.85.017 0.5984 19.47 6.595 1.137 1.962 11.77 XPA.85.287 0.09091 1.8650.3042 0.2808 0.2061 1.515 XPA.85.339 0.09841 2.984 0.4791 0.2575 0.327318.68 XPA.85.341 0.1956 5.387 1.109 0.3371 0.7778 14.46

F. Binding of Affinity Matured XPA.85.012 Variants to CHOK1, CHO HumanPTH1R, CHO Mouse PTH1R, UMR106, and Saos-2 Cells

To characterize antibody binding to PTH1R, cells were labeled witheither CellTrace™ CFSE or CellTrace™ Violet. Unlabeled human PTH1R,CHOK1 and Saos-2 labeled with CellTrace™CFSE, and CHO mouse PTH1R andUMR106 labeled with CellTrace™Violet were pooled together and stainedwith increasing concentrations of antibody. Antibodies were incubatedfor 1 hr at 4° C. followed by two washes with FACS Buffer (0.5% BSA,0.1% NaN3 in DPBS). Cells were then incubated with Allophycocyanin (APC)AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG (H+L) (JacksonImmunoResearch, West Grove, Pa.) for 20 minutes at 4° C. Followed by twowashes, cells were resuspended in FACS Buffer. Samples were acquiredusing BD Cytometer (BD Biosciences, San Jose, Calif.) and data wereanalyzed using FlowJo (FlowJo, LLC, Ashland, Oreg.) and Prism (GraphPadSoftware, La Jolla, Calif.). All antibodies recognized PTH1R expressedon CHO human PTH1R, CHO mouse PTH1R, and UMR106. These antibodies alsodo not bind nonspecifically to CHOK1 parent and Saos-2 cells (Table 5).

TABLE 5 FACS Binding Profiles of XPA.85.012 Variants. CHO Human CHOMouse Antibody CHOK1 PTH1R PTH1R UMR106 XPA.85.288 − + + + XPA.85.326− + + + XPA.85.327 − + + + XPA.85.331 − + + + XPA.85.332 − + + +XPA.85.333 − + + + XPA.85.334 − + + + XPA.85.342 − + + + XPA.85.343− + + + XPA.85.344 − + + + XPA.85.345 − + + + XPA.85.346 − + + +XPA.85.347 − + + + (+) indicates binding and (−) indicates no binding.

G. Antibody Binding Specificity to Human PTH1R on Saos-2 OverexpressingHuman PTH1R Cells

To confirm specificity of the anti-PTH1R antibodies, antibodies werescreened against Saos-2 overexpressing human PTH1R cells in the presenceor absence of both ligands, PTH or PTHrP (FIG. 7A-7D).

H. Assessment of PTH2R Cross-Reactivity Using CHO Overexpressing HumanPTH2R Cells

Anti-PTH1R antibodies (XPA.85.328, XPA.85.329, and XPA.85.330) werescreened against CHO cells overexpressing human PTH2R to determinereceptor cross-reactivity (FIG. 8). PTH2R has a 51% homology to PTH1R.To confirm anti-PTH1R antibodies binding specificity to PTH1R only,increasing concentration of antibodies were incubated with CHO humanPTH2R for 1 hr at 4° C. Following two washes, cells were incubated withAllophycocyanin (APC) AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG(H+L) (Jackson ImmunoResearch, West Grove, Pa.) for 20 minutes at 4° C.Cells were washed twice and resuspended in FACS Buffer. Samples wereacquired using BD Cytometer (BD Biosciences, San Jose, Calif.) and datawere analyzed using FlowJo (FlowJo, LLC, Ashland, Oreg.) and Prism(GraphPad Software, La Jolla, Calif.). As shown in FIG. 8, allantibodies showed no binding to CHO human PTH2R (i.e. nocross-reactivity to PTH2R) and are specific to PTH1R. Anti-KLH.G2 wasused a negative control for binding.

Example 5: Functional Assays

A. cAMP Accumulation Assays

In most (if not all) cell types, activation of PTH1R results instimulation of adenylyl cyclase and accumulation of intracellular cAMPvia coupling to Gs, followed by stimulation of protein kinase A (PKA).The functional activity of purified, reformatted IgGs was routinelytested by measuring their ability to antagonize PTH1R-mediated cAMPaccumulation via inhibition of the Gs/PKA pathway

Human PTH1R CHOK1 Clone 22 and mouse PTH1R CHOK1 cells were grown inExCell 302 Serum-Free Media for CHO Cells (Sigma, St. Louis, Mo.)containing 4 mM L-Glutamine and 0.4 mg/ml Geneticin (Life Technologies;ThermoFisher Scientific, Waltham, Mass.). On the day of the experiment,cells were harvested by centrifugation (1500 rpm for 3 min at roomtemperature). Growth media was gently aspirated, and cells were washedonce in Dulbecco's Phosphate Buffered Saline, No Calcium or Magnesium(DPBS; Life Technologies; ThermoFisher Scientific, Waltham, Mass.).Cells were then spun down a second time at 1500 rpm for 3 min at roomtemperature. Wash solution was gently aspirated and cells wereresuspended in assay buffer [DPBS containing 0.1% BSA and 1 mM3-Isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, St. Louis, Mo.)].Cells were counted using a Vi-CELL (Beckman Coulter, Indianapolis,Ind.), and adjusted to a density of 2.5×1E⁶ viable cells/ml or 50,000cells/well using assay buffer. 20 μl/well of cells were dispensed to96-well, white, flat bottom, tissue culture treated assay plates(Corning). Cells were preincubated with 5 μl of 6×IgG for 30 min at 37°C. Following preincubation, cAMP accumulation was induced for 45 min at37° C. by the addition of 5 μl of 6× ligand [(either PTH(1-34) orPTHrP(1-34)]. cAMP accumulation was measured using a HitHunter cAMP forBiologics kit (DiscoveRx, Fremont, Calif.) per manufacturer'sinstructions. Human PTH(1-34) was purchased from Bio-Techne(Minneapolis, Minn.) and human PTHrP(1-34) was obtained fromSigma-Aldrich (St. Louis, Mo.). Stock solutions of both peptides wereprepared in DPBS containing 0.1% BSA, aliquotted and stored at −80° C.Ligand and IgG dilutions were made using DPBS containing 0.1% BSA and 1mM IBMX. Chemiluminescent signal was measured using a FlexStation 3(Molecular Devices, Sunnyvale, Calif.). Data are expressed as relativeluminescence units (RLUs). Curve-fitting was carried out using GraphPadPrism 6.0 (GraphPad Software Inc., San Diego, Calif.).

Human PTH1R CHOK1 Clone 22 cells were preincubated for 30 min at 37° C.with 267 nM (40 μg/ml) of either XPA.85.012 (solid squares) orXPA.85.017 (solid triangles), followed by induction with increasingconcentrations of either PTH(1-34) (FIG. 9A) or PTHrP(1-34) (FIG. 9B).Both antibodies inhibited ligand-mediated cAMP accumulation (Gs/PKApathway).

Following affinity engineering of XPA.85.017 (open circles) by lightchain shuffling, CHOK1 cells stably overexpressing either human (FIG.10A and FIG. 10B) or mouse PTH1R (FIG. 10C and FIG. 10D) werepreincubated for 30 min at 37° C. with 267 nM (40 μg/ml) of variantIgGs, followed by induction with increasing concentrations of PTH(1-34)for 45 min at 37° C. Variant IgGs exhibited a range of inhibition ofligand-mediated cAMP accumulation (Gs/PKA pathway) at human and mousePTH1R.

SaOS-2 and UMR106 cells were obtained from ATCC (Manassas, Va.). SaOS-2cells were grown in McCoy's 5A (Modified) Medium (Life Technologies;ThermoFisher Scientific, Waltham, Mass.) containing 15% heat-inactivatedfetal bovine serum (FBS) (Hyclone; ThermoFisher Scientific, Waltham,Mass.). UMR106 cells were grown in Dulbecco's Modified Eagle Mediumcontaining 4.5 g/L D-glucose, supplemented with 10% heat-inactivatedFBS, 1 mM sodium pyruvate (Life Technologies; ThermoFisher Scientific,Waltham, Mass.), and 2 mM L-Glutamine. SaOS-2 cells were seeded in96-well assay plates at a density of 50,000 cells/well for 3 days, thenserum starved overnight in medium containing no FBS but supplementedwith 1% BSA. UMR106 cells were seeded in 96-well assay plates at adensity of 35,000 cells/well for 1 day, then serum starved overnight inmedium containing no FBS but supplemented with 1% BSA. Antagonism ofcAMP accumulation by IgGs was measured as described previously for CHOK1cell lines.

Affinity engineering of XPA.85.017 by light chain shuffling resulted inXPA.85.287. This variant showed a significant improvement in activityagainst human PTH1R (FIG. 11B, open squares) vs. parent (solid squares),with little change in activity against rat PTH1R (FIG. 11C). Incontrast, affinity engineering of XPA.85.012 (solid triangles) resultedin a variant, XPA.85.288 (open triangles), showing a significantimprovement in activity against both human PTH1R (data not shown) aswell as rat PTH1R. The XPA.85.288 was also found to significantlyinhibit PTHrP-induced cAMP in SaOS-2 Cells Expressing Native Human PTH1R(FIG. 11D).

Concentration-response curves were performed for PTHrP(1-34) andincreasing concentrations of XPA.85.287 (an affinity engineered variantof XPA.85.017) in cAMP assays using human PTH1R CHOK1 (FIG. 12A) andmouse PTH1R CHOK1 (FIG. 12B) cells. Schild regression analyses wereperformed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego,Calif.), revealing non-parallel rightward shifts inconcentration-response curves. These results are consistent with anon-competitive (allosteric) mechanism of action of the variant IgG.

Concentration-response curves were performed for PTHrP(1-34) andincreasing concentrations of XPA.85.288 (an affinity engineered variantof XPA.85.012) in cAMP assays using human PTH1R CHOK1 (FIG. 13A) andmouse PTH1R CHOK1 (FIG. 13B) cells. Schild regression analyses wereperformed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego,Calif.), revealing non-parallel rightward shifts inconcentration-response curves. These results are consistent with anon-competitive (allosteric) mechanism of action of the variant IgG.

B. Calcium Mobilization Assays

Activation of PTH1R in some cell types leads to Gq coupling, activationof phospholipase CP and hydrolysis of membrane-associatedphosphatidylinositol to form inositol phosphates, increases inintracellular calcium, and activation of calcium-dependent enzymes,including protein kinase C (PKC). The functional activity of purified,reformatted anti-PTH1R IgGs was tested by measuring their ability toantagonize PTH1R-mediated calcium mobilization via inhibition of theGq/PKC pathway.

Human PTH1R CHOK1 Clone 22 and mouse PTH1R CHOK1 cells were grown inExCell 302 Serum-Free Media for CHO Cells (Sigma, St. Louis, Mo.)containing 4 mM L-Glutamine and 0.4 mg/ml Geneticin (Life Technologies,Waltham, Mass.). Cells were seeded at 35,000 cells/well in 100 μl/wellof growth media on 96-well, black/clear bottom, tissue culture treated,Poly-D-Lysine coated assay plates (Corning BioCoat; Corning, N.Y.) andincubated overnight at 37° C. [5% CO₂, 95% relative humidity]. The nextday, 100 μl/well of 2× Calcium 5 loading dye (Molecular Devices,Sunnyvale, Calif.) containing 5 mM probenecid (Sigma-Aldrich, St. Louis,Mo.) was prepared according to manufacturer's instructions and added,and plates were incubated for 30 min at 37° C. Following incubation, 25μl of loading dye/growth media was manually removed from each well,followed by addition of 25 μl of 10× test IgG (antibody dilutions wereprepared in 1×HBSS containing 20 mM HEPES). Plates were then returned tothe incubator for an additional 30 min at 37° C. After that, assayplates were centrifuged at 1500 rpm for 3 min at room temperature.Plates were then placed in a FlexStation 3 (Molecular Devices,Sunnyvale, Calif.) prewarmed to 37° C. to equilibrate for 5 min prior toassay. Basal fluorescence was recorded every second for 19 seconds priorto the addition of 50 μl of 5× ligand (either PTH(1-34) or PTHrP(1-34)).Fluorescence was recorded every second for 1 min, then every 6 secondsfor an additional 2 min. Data was expressed as “Max-Min”, andcurve-fitting was carried out using GraphPad Prism 6.0 (GraphPadSoftware Inc., San Diego, Calif.). Human PTH(1-34) was purchased fromBio-Techne (Minneapolis, Minn.) and human PTHrP(1-34) was obtained fromSigma-Aldrich (St. Louis, Mo.). Stock solutions of both peptides wereprepared in DPBS containing 0.1% BSA, aliquotted and stored at −80° C.

Human PTH1R CHOK1 Clone 22 cells were preincubated for 30 min at 37° C.with 267 nM (40 μg/ml) of either XPA.85.012 (solid squares) orXPA.85.017 (solid triangles), followed by induction with increasingconcentrations of either PTH(1-34) (FIG. 9C) or PTHrP(1-34) (FIG. 9D).Both antibodies inhibited ligand-mediated calcium mobilization (Gs/PKApathway).

TABLE 6 EC50 values in pM for PTH(1-34) and PTHrP(1-34) in the presenceof variant IgGs derived from affinity engineering of XPA.85.017 (parent)by light chain shuffling cAMP Assay cAMP Assay cAMP Assay cAMP AssaySaOS-2 (Endogenous UMR106 (Endogenous Human PTH1R CHOK1 Mouse PTH1RCHOK1 Human PTH1R) Rat PTH1R) Ligand EC50 (pM) Ligand EC50 (pM) LigandEC50 (pM) Ligand EC50 (pM) Ligand PTH(1-34) PTHrP(1-34) PTH(1-34)PTHrP(1-34) PTH(1-34) PTHrP(1-34) PTH(1-34) PTHrP(1-34) Vehicle Only30.7 ± 61.4 ± 30.6 ± 43.1 ± 524.5 ± 698.7 ± 646.6 ± 333.2 ± 7.7 (10)18.1 (3) 10.8 (5) 17.8 (3) 130.5 (6) 128.8 (4) 210.6 (8) 68.3 (6)XPA.85.017 140.6 ± 967.6 ± 2811.0 ± 3742.3 ± 797.8 ± ND 23820 ± 7776.7 ±(Parent) 49.9 (4) 491.0 (3) 1097.1 (3) 1288.1 (3) 202.4 (2) 5968.1 (4)1052.0 (3) 5-fold shift 16-fold shift 91-fold shift 87-fold shift Noshift 37-fold shift 23-fold shift XPA.85.287 908.3 ± 52700 13640 576678600 ± 62200 ± 66700 ± 11590 (1) 341.3 (4) (1) >100- (1) >100-(1) >100- 18179 21400 (3) 16628 35-fold shift 29-fold shift fold shiftfold shift fold shift (3) >100- 89-fold shift (3) >100- fold shift foldshift XPA.85.335 42.9 ± ND 4545 (1) ND ND  6412 (1) ND ND 5.3 (2)XPA.85.336 18.1 ± ND 144.4 (1)  ND ND 23580 (1) ND ND 8.4 (2) XPA.85.337107.2 ± ND 219.4 (1)  ND ND 27080 (1) 2877 (1) ND 44.9 (2) XPA.85.338244.5 ± ND 1802 (1) ND 32790 (1) 32950 (1) 7861 (1) ND 28.9 (2)XPA.85.339 612.8 ± ND 1952 (1) ND 24768 ± 31450 (1) 7900 ±  3481 (1)140.6 (2) 10586 (3) 21 (2) XPA.85.340 403.5 ± ND 614.2 (1)  ND  4169 (1)11850 (1) 8100 (1) ND 15.9 (2) XPA.85.341 403.9 ± ND 2147 (1) ND 38100(1) 62990 (1) 13740 (1)  ND 175.5 (2) Variant IgGs were tested at 267 nM(40 μg/ml), and were preincubated with cells for 30 min at 37° C. priorto induction for 45 min at 37° C. Fold-shift in ligand EC50 wascalculated relative to the EC50 of ligand in the “Vehicle Only” controlgroup. Number of experiments is indicated in parentheses. ND = NotDetermined.

TABLE 7 Percent inhibition of cAMP accumulation mediated by 1 μM ofeither PTH(1-34) or PTHrP(1-34) in the presence of variant IgGs derivedfrom affinity engineering of XPA.85.012 (parent) by light chainshuffling. cAMP Assay SaOS-2 cAMP Assay UMR106 (Endogenous Human PTH1R)(Endogenous Rat PTH1R) % Inhibition at 1 % Inhibition at 1 μM of ligandμM of ligand Ligand PTH(1-34) PTHrP(1-34) PTH(1-34) PTHrP(1-34)XPA.85.012 ND  0 (1)  0 (1)  0 (1) (Parent) XPA.85.288 79 (2) 79 (1) 73(2) 45 (1) XPA.85.329 79 (1) 90 (1) 50 (1) 41 (1) XPA.85.330 85 (1) 89(1) 55 (1) 56 (1) XPA.85.331 67 (1) 72 (1) 42 (1) 37 (1) XPA.85.332 NDND ND ND XPA.85.333 ND ND ND ND XPA.85.334 ND ND ND ND XPA.85.342  0 (2)17 (1)  6 (1)  0 (1) XPA.85.343 ND ND ND ND XPA.85.344 ND ND ND NDXPA.85.345 ND ND ND ND XPA.85.346 72 (1) 75 (1) 40 (1) 31 (1) XPA.85.347ND ND ND ND Number of experiments is indicated in parentheses ND = NotDetermined Variant IgGs were tested at 267 nM or 40 μg/ml, and werepreincubated with cells for 30 min at 37° C. prior to induction of cAMPaccumulation for 45 min at 37° C. Data represent average RLUs induplicate wells.

TABLE 8 IC50 values of variant IgGs derived from affinity engineering ofXPA.85.012 (parent) by light chain shuffling. cAMP Assay cAMP Assay cAMPAssay cAMP Assay Human Human Mouse Mouse PTH1R PTH1R PTH1R PTH1R CHOK1CHOK1 CHOK1 CHOK1 Antibody PTHrP(1-34) PTH(1-34) PTHrP(1-34) PTH(1-34)Name IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) XPA.85.012 ND ND ND ND(Parent) XPA.85.328 7.06 11.82 2.23 ND XPA.85.329 6.54 16.55 2.66 NDXPA.85.330 8.23 10.6 2.82 ND XPA.85.331 8.86 14.21 3.2 ND XPA.85.3325.03 12.1 2.3 ND XPA.85.333 5.61 11.43 2.04 ND XPA.85.334 12.39 51.19*3.32 ND XPA.85.342 ND ND ND ND XPA.85.343 17.77 14.38 3.87 ND XPA.85.34415.28 15.53 3.89 ND XPA.85.345 12.14 14.19 2.53 ND XPA.85.346 14.5729.85* 3.57 ND XPA.85.347 Partial Partial 2.9 ND *Curve does not reach aplateau ND = Not Determined Variant IgGs were tested at a range ofconcentrations (0-66.67 nM or 10 μg/ml), and were preincubated withcells for 30 min at 37° C. prior to induction of cAMP accumulation byeither PTH(1-34) or PTHrP(1-34) for 45 min at 37° C. Data representaverage RLUs in duplicate wells. Variants exhibited a range ofantagonism of ligand-mediated cAMP accumulation, particularly againstthe human PTH1R.

C. M-CSF Secretion by Saos-2 Osteoblasts

PTH and PTHrP act on the PTH1R receptor expressed in osteoblasts andosteocytes to stimulate the production of macrophage colony stimulatingfactor (M-CSF) and the surface expression Receptor activator of nuclearfactor kappa-B ligand (RANKL). The increased production of M-CSF andRANKL expression by these bone cells drives the differentiation andactivation of bone resorbing osteoclasts, which leads to increased levelof calcium efflux from the bone and bone demineralization. Both of thesegrowth factors are required for the PTH/PTHrP mediated differentiationand activation of human osteoclasts (Matsuzaki et al., 1999Endocrinology 140:925-32; Itoh et al., 2000 Journal of bone and mineralresearch: the official journal of the American Society for Bone andMineral Research 15:1766-75).

To assess the ability of these antibodies inhibit the PTH or PTHrPstimulated secretion of osteoclastogenic growth factors, an M-CSFsecretion experiment using the osteoblastic cell line Saos-2 wasemployed.

Saos-2 cells (ATCC, Manassas Va.) were grown at 37° C. with 5% CO₂ andsaturating humidity in 15% McCoy's 5A medium (Life Technologies) with15% FBS (Hyclone/GE Life Sciences, Logan, Utah) plated at 50,000cells/well into a 96 well culture plate in growth media and allowed toform a confluent monolayer for 48 hours. Then the media was replacedwith MEM-α (Life Technologies) with 10% FBS and incubated for anadditional 72 to 96 hours. The media was aspirated from the wells andreplaced with 2× antibody treatments in the same MEM-α based media asabove. The antibody treatments were incubated at 37° C. for 20 to 30minutes prior to the addition of the PTH (1-34) or PTHrP (1-36) peptidedilutions in media. The ligands were diluted to achieve the shown finalconcentration following addition to the assay well. For ligand titrationexperiments the final antibody concentration is 40 μm/mL (267 nM). Theplate was returned to the incubator and incubated at 37° C. for 48hours. The supernatant from the treatment wells were then analyzed by aplate based immuno-assay which measures human M-CSF (MesoScaleDiagnostics, Rockville Md.) following the manufacturer's instructionsand read on a Sector Imager 6000 (MesoScale Diagnostics). For theantibody titration experiment, the same protocol was followed exceptthat a fixed concentration of ligand (12.5 nM PTHrP (1-36)) was used andthe concentration of antibody was varied.

The affinity matured antibodies demonstrated the ability to inhibit thePTH stimulated increases in M-CSF secretion by Saos-2 cells. Theantibody XPA.85.287 shifted the PTH (1-34) dose response curve byapproximately 7 fold (1.2 nM to 8.7 nM) (FIG. 14A). In a separateexperiment, the light chain variants XPA.85.288, XPA.85.346, XPA.85.331,and XPA.327 all shift the PTH (1-34) dose response curve greater than 10fold (FIG. 14B).

Affinity matured light chain variants also inhibited PTHrP mediatedincreases in M-CSF secretion by Saos-2 cells. At a fixed antibodyconcentration of 40m/mL, XPA.85.328, XPA.329, and XPA.85.330 inhibitPTHrP (1-36) stimulated M-CSF secretion dose response by greater than200 fold (FIG. 15A). In a separate experiment, the parental clonesXPA.85.012 and XPA.85.017 were compared to their affinity maturedvariants XPA.85.288 and XPA.85.287 respectively. The affinity maturedclones showed significant inhibition of PTHrP induced M-CSF secretionwhereas the parental clones did not (FIG. 15C). The antibody doseresponse of this effect was also evaluated against a fixed PTHrP (1-36)concentration of 12.5 nM. XPA.85.328, XPA.329, and XPA.85.330 fullyneutralized M-CSF production induced by the 12.5 nM PTHrP treatment withEC50s of 6.6 nM, 4.0 nM, and 1.5 nM respectively, (FIG. 15B).

D. ERK Phosphorylation

The MAPK (mitogen-activated protein kinase) ERK1/2 (extracellularsignal-regulated kinase) is phosphorylated and activated followingligand stimulation of the PTH1R. This pathway activation isrepresentative of a convergent signaling event requiring inputs fromboth the G-protein mediated adenylate cyclase pathway and the β-Arrestinmediated pathway and has been shown to be required for renal calciumtransport (Gesty-Palmer et al.,2006 JBC 281:10856-64; Sneddon et al.,2000 Endocrinology 141:4185-93).

In order to determine the ability of the anti-PTH1R antibodies tomodulate the MAPK pathway, experiments were carried out which evaluatedtheir ability to inhibit PTH and PTHrP mediated ERK1/2 phosphorylationat Thr202/Tyr204; Thr185/Tyr187.

CHO-PTH1R cells were serum starved at 1×10⁶ cells/mL in RPMI 1640 (LifeTechnologies) with 0.5% BSA (Sigma), and incubated overnight at on ashaking platform at 37° C. and 5% CO₂. The next day, cells were seededat a density of 100,000 cells per well in assay buffer (PBS+0.5% BSA)into 96-well U-bottom plates. Subsequently, 50 μL per well of antibodiesdiluted to 400 nM (2×-60 μg/mL) in assay buffer was added to cells.After 10 minutes of incubation at 37° C. 25 μL/well of the dilutedLigands (4× of final concentration) were added to the wells containingthe cells and antibodies, and incubated for 5 minutes at 37° C. Then,100 μl of cold PBS per well was added to stop the reaction. The platewas centrifuged for 3 minutes at 4° C. and the media was removed. Then80 ul of ice cold MSD lysis buffer was added to cells and resuspended.The plate was incubated on a shaking platform for 1 hour at 4° C. tocompletely lyse cells. The total and phosphorylated ERK were measuredusing an MSD whole cell lysate Phospho-ERK1/2 kit (MesoScale Diagnosticscat. K11107D-1) as per the manufacturer's instructions and read on aSector Imager 6000 (MesoScale Diagnostics). Curves were fit by nonlinearregression using the sigmoidal dose-response equation in GraphPad Prismversion 6.05 (GraphPad, San Diego, Calif.).

The antibody XPA.85.012 increased the EC50 of PTH (1-34) induced ERK1/2phosphorylation by approximately 9 fold (3.8 nM to 34.6 nM) (FIG. 16A)and the EC50 of PTHrP (1-34) induced ERK1/2 phosphorylation increasedroughly 6 fold (EC50 of 10.6 nM to 62.5 nM) (FIG. 16B). The antibodyXPA.85.017 increased the EC50 of PTH (1-34) induced ERK phosphorylationby greater than 100 fold (3.8 nM to >500 nM) (FIG. 16A) and the EC50 ofPTHrP (1-34) increased roughly 80 fold (EC50 of 10.6 nM to 887 nM) (FIG.16B).

Example 6: Manufacturability Improvements

A. Evaluation of V Region Sequences for Selected Antibody Candidates

The light and heavy chain V region amino acid sequences of selectedantibody candidates were evaluated for the presence of “non-human” aminoacids using Human Engineering software (HE™, U.S. Pat. No. 5,766,886)and for N-linked glycan, oxidation and deamidation signals usingbioinformatics software. Based on these analyses for certain antibodies,amino acid changes were incorporated by site directed mutagenesis orgene synthesis.

B. CHO-K1 Manufacturability Assessment of Selected Antibody Candidates

Transient expression in CHO-K1 cells was used to evaluatemanufacturability of multiple antibody.

DNAs encoding the light and heavy chain V regions were cloned intotransient expression plasmids containing antibody signal sequences andlight chain (LC) or heavy chain (HC) constant regions, respectively andtransfected into suspension-adapted CHO-K1 cells in deep well 96 wellplates. Expression plasmids were tested at LC:HC plasmid ratios (byweight) of 1:1, 2:1, 4:1 and 1:2 with the DNA quantity in eachtransfection kept constant by the addition of empty plasmid DNA.Duplicate transfections are performed for each antibody. Two controlantibodies, XB10 and XE17, are included as controls for high and lowexpression, respectively. Following transfection, plates were incubatedon a shaker platform in a CO₂ incubator for 7 days with a temperatureshift from 37° C. to 32° C. 24 hr post-transfection. Supernatants wereassayed for antibody titer using an Octet instrument with Protein Asensor tips.

Selected antibody candidates were evaluated for expression using thisassay. Based on the results observed for some of the antibodies, the DNAsequences encoding the HC and LC V regions along with the signalsequences were optimized for expression in CHO cells (GeneArt). Theoptimized antibodies then were re-tested for manufacturability. In caseswhere expression optimization was carried out, while the DNA sequenceswere different, the amino acid sequences for the light and heavy chain Vregions remained the same unless Human Engineering analysis indicatedthe need for an amino acid change. The antibodies with optimized DNAsequences are shown in Table 9B.

TABLE 9A XPA.85.287, XPA.85.339, XPA.85.340 and XPA.85.341 HC:LC Titerrelative to XB10 (average result from duplicate transfections) RatioXB10 XE17 XPA.85.287 XPA.85.339 XPA.85.340 XPA.85.341 1:1 100 21 49 5077 71 1:2 51 15 97 60 83 74 1:4 17 8 47 16 62 19 2:1 40 6 16 16 21 17

TABLE 9B XPA.85.288, XPA.85.326 and XPA.85.327 and their expression-optimized versions, XPA.85.328, XPA.85.329 and XPA.85.330. Titerrelative to XB10 (average result from duplicate transfections) HC:LCOriginal Optimized Original Optimized Original Optimized Ratio XB10 XE17XPA.85.288 XPA.85.328 XPA.85.326 XPA.85.329 XPA.85.327 XPA.85.330 1:1100 26 7 78 25 87 19 125 1:2 55 30 14 59 86 91 75 101 1:4 39 21 7 26 3737 49 58 2:1 84 9 1 23 7 24 4 16

TABLE 9C XPA.85.331, XPA.85.332, XPA.85.333 and XPA.85.334 HC:LC Titerrelative to XB10 (average result from duplicate transfections) RatioNXB10 NXE17 (XPA.85.331) (XPA.85.332) (XPA.85.333) (XPA.85.334) 1:1 10029 33 28 37 19 1:2 71 20 75 68 98 28 1:4 13 8 26 37 42 9 2:1 40 8 6 5 62

Table 9. Results of CHO-K1 Manufacturability Assessment ofAffinity-Matured Anti-PTH1R Antibodies.

Values reported are relative to expression of the “high-expressing”control antibody, XB10, set to 100 at a 1:1 LC:HC ratio. Highestexpression is highlighted in bold. Expression optimization was performedfor three of the antibodies.

For the affinity-matured antibodies XPA.85.287, XPA.85.339. XPA.85.340and XPA.85.341, highest expression was observed at a HC:LC ratio of 1:2(Table 9A). Although expression was lower at a HC:LC ratio of 1:1 thanat 1:2, the level achieved at a 1:1 ratio is acceptable for cell linedevelopment. Three other affinity-matured antibodies, XPA.85.288,XPA.85.326 and XPA.85.327 displayed very low expression at a 1:1 HC:LCratio. A significant improvement in expression was observed for allthree of these antibodies especially at HC:LC ratio of 1:1 afterexpression optimization of the V region DNA sequences. XPA.58.328(optimized version of XPA.85.288) displayed >10-fold increase vs.XPA.85.288. XPA.85.329 (optimized version of XPA.85.326) displayed a˜3.5-fold increase vs. XPA.85.326 and XPA.85.330 (optimized version ofXPA.85.327) displayed ˜6.5-fold increase vs. XPA.85.327 (Table 9B).

Four additional affinity-matured antibodies, XPA.85.331, XPA.85.332,XPA.85.333 and XPA.85.324 also were evaluated for CHO manufacturability.All four antibodies displayed low expression at a HC:LC ratio of 1:1being ≦than the poor-expressing XE17 antibody (Table 9C). Expression was2-2.5-fold higher at HC:LC ratios of 1:2. XPA.85.334 expression fellbelow the minimum acceptable level in this assay to qualify forinitiating cell line development without further expression improvement.

Example 7: Measurement of the In Vivo Effects of Anti-PTH1R ReceptorAntibodies A. Thyroparathyroidextomized (TPTx) Model

Anti-human PTI-11R receptor antibodies found to be cross-reactive withrat PTH1R were tested in in vivo models of primary hyperparathyroidism(for PTH) and Humoral Hypercalcemia of Malignancy (HHM for PTHrP). In athyroparathyroidectomized (TPTx) model, the effects of the endogenouscalciotropic peptide hormones, PTH and calcitonin are eliminated bysurgical removal of the thyroid and parathyroid glands in Sprague-Dawley(SD) male rats (Charles River Laboratories, Raleigh). Low postoperativeserum calcium levels are then obtained in a tightly controlled setting.Thyroid pellets (L-thyroxine T4, 0.25 mg; 60 days slow release;Innovative. Research of America) are implanted subcutaneously underanesthesia. The jugular vein is then cannulated for continuous infusionof PTH(1-34) (1.25 μg/kg/hr at a rate of 1 mL/kg/hr for 6 hr. This modelwas used to evaluate the ability of PTH1R antibodies to affect theincrease of serum calcium levels induced by PTH(1-34) intravenousinfusion.

To determine whether anti-PTH1R receptor antibodies reducePTH(1-34)-induced elevation of serum calcium levels, Sprague-Dawley (SD)male rats (n=5-6/group) were challenged intravenously with antibodiesXPA.85.017 (Ab017), XPA.85.287 (Ab287), XPA.85.288 (Ab288) and BM2 (ananti-PTH antibody), or an isotype control (15 mg/kg), 18 hr beforeinitiation of PTH(1-34) infusion (FIG. 17).

The anti-PTH antibody was based on Abgenix's antibody ID#183 (U.S. Pat.No. 7,318,925 B2. The heavy (HC) and light chain (LC) V region sequenceswere expression optimized for production in CHO cells and synthesizedwith appropriate restriction sites for cloning (GeneArt). DNAs werecloned into transient expression plasmids containing signal sequencesand LC or HC constant regions. Each HC or LC is under control of thehuman CMV (hCMV) promoter and mouse light chain 3′ untranslated region.The expression plasmids also contain the Epstein-Barr virus origin ofreplication to allow replication in cell lines containing theEpstein-Barr virus nuclear antigen. Transient expression using theExpi293 expression system (Life Technologies, Carlsbad, Calif.) was usedto generate anti-PTH antibody for in vitro and in vivo studies. Antibodypurification was carried out at Aragen Bioscience (Morgan Hill, Calif.).

Serum calcium was measured before dosing (baseline), pre-infusion (T0),2, 4 and 6 hr post start of infusion. Over the first 4 hr infusion, thetotal calcium level in the isotype group increased. In rats receivingAb287, the calcium level was comparable to the levels of the isotypecontrol group. Calcium levels of rats receiving Ab017 slightlydecreased. Compared to isotype controls, rats receiving BM2 and Ab288had significantly lower levels of calcium throughout the infusion.

B. hPTH(1-34) Infusion Model

To further evaluate the effect of anti-PTH1R receptor antibodies oncalcemic control, hPTH(1-34) was continuously infused subcutaneously(Alzet mini pump, 2ML1; 100/hr, 10 μg/kg/day) in normal Sprague Dawleyrats (Harlan) for 7 days to mimic PTH hypersecretion in patients withhyperparathyroidism. Calcium was measured as biomarker to assess in vivoneutralization by a single intravenous administration of Ab288 (2 or 10mg/kg; n=5/group), BM2 (10 mg/kg; n=5), or isotype control (10 mg/kg,n=2) 24 hr after pump implantation (FIG. 18).

Serum calcium was measured before pump implantation (Predose), 24, 27 (3hr post dose), 48, 72, 96, 120, 144 and 168 hours post pumpimplantation. Over the first 72 hour infusion, the total calcium levelin the isotype group increased. Compared to isotype controls, ratsreceiving BM2 and Ab288 (2 and 10 mg/kg) had significantly lower levelsof calcium 24 hr post dose and throughout the infusion: the effect onserum calcium levels of 2 mg/kg Ab288 was comparable to that of 10 mg/kgBM2. 10 mg/kg Ab288 produced a dramatic hypocalcemic effect (FIG. 18A).

Antibodies, Ab328, Ab329 and Ab330 (2 mg/kg IV) were tested in a similarstudy where serum calcium was measured before pump implantation(Predose), 24, 27 (3 hr post dose), 48, 72, 96 and 120 hours post pumpimplantation. All antibodies significantly lowered levels of calcium 24hr post dose and throughout the infusion, with Ab288 and Ab328 producingthe most dramatic reduction in total serum calcium levels (FIG. 18B).

C. hPTHrP(1-36) Infusion Model

Similarly, the effects of anti-PTH1R receptor antibodies on inhibitionof PTHrP activity were assessed by continuously infusing hPTHrP(1-36)subcutaneously (Alzet mini pump, 2ML1; 10 μl/hr, 100 mg/kg/day) innormal Sprague Dawley rats (Harlan) for 6 days to mimic hypercalcemia inpatients with hyperparathyroidism. Calcium was measured as a biomarkerto assess in vivo neutralization by a single intravenous administrationof Ab288 (2 or 10 mg/kg; n=4/group), anti-PTHrP antibody MCB1.1 (10mg/kg; n=5), or isotype control (10 mg/kg, n=3) 24 hr after pumpimplantation.

The anti-PTHrP antibody heavy chain and light chain V region sequenceswere based on Onuma et al., Anticancer Research 24:2665-2674, 2004. Theheavy chain (HC) and light chain (LC) V region sequences were expressionoptimized for production in CHO cells and synthesized with theappropriate restriction sites (GeneArt) for cloning into transientexpression plasmids containing signal sequences and LC or HC constantregions. Each HC or LC is under control of the human CMV (hCMV) promoterand mouse light chain 3′ untranslated region. The expression plasmidsalso contain the Epstein-Barr virus origin of replication to allowreplication in cell lines containing the Epstein-Barr virus nuclearantigen. Transient expression using the Expi293 expression system (LifeTechnologies, Carlsbad, Calif.) was used to generate anti-PTHrP antibodyfor in vitro and in vivo studies. Antibody purification was carried outat Aragen Bioscience (Morgan Hill, Calif.).

Serum calcium was measured before pump implantation (Predose), 24, 26 (2hr post dose), 48, 72, 96, 120 and 144 hours post pump implantation(FIG. 19A). Body weights were measured before pump implantation(Predose), 26 (2 hr post dose), 48, 96 and 144 hours post pumpimplantation (FIG. 19B).

Over the first 48 hour infusion, the total calcium level in the isotypegroup increased. Compared to isotype controls, rats receiving MCB1.1 andAb288 (2 and 10 mg/kg) had significantly lower levels of calcium 24 hrpost dose. These lower calcium levels persisted throughout the infusionfor Ab288 (2 and 10 mg/kg). The effect on serum calcium levels of 2mg/kg Ab288 was more pronounced to that of 10 mg/kg MCB1.1. Ab288 (10mg/kg) produced a dramatic hypocalcemic effect. PTHrP(1-36) infusioninduced weight loss that was prevented by Ab288 (2 and 10 mg/kg).

This suggests anti-PTH1R antibodies could have health benefits andpotentially prevent cachexia, a wasting disorder of adipose and skeletalmuscle tissues that leads to profound weight loss.

Pilot studies had shown that infusion of doses of PTH and PTHrP canproduce levels of serum calcium higher than 14 mg/dL linked tomortality. Treatment with anti-PTH1R antibody could preventhypercalcemia (>14 mg/dL) and could therefore prevent mortality in HHM(survival benefit).

D. Tumor Model

To determine whether anti-PTH1R receptor antibodies reduce tumor-inducedelevation of serum calcium levels, 6 week old CDF1 female mice, orAthymic Nude (NCr-nu/nu) mice were challenged intravenously withantibody XPA.85.328 (Ab328) (10 mg/kg), after serum calcium was foundelevated in three different tumor models (mouse colon C26, humanprostate PC-3 and human lung cancer HARA-B, respectively). Therespective cells were cultured, and when the needed number of cells wasobtained, each mouse were injected SC in the right flank with fivemillion (5×10⁶) C26 cells, 10 million (1×10⁷) PC-3 cells or 10 million(1×10⁷) HARA-B cells in 0.1 mL of serum-free media. Serum calcium wasmeasured regularly until hypercalcemia was observed (>12 mg/dL), usuallywhen tumors were >1000 mg. Mice were then treated with Ab328 10 mg/kgiv, and in the HARA-B model, a separate group of mice with tumors >1 gwas treated with the isotype (negative control; anti KLH G2) at 10 mg/kgiv, and the total calcium levels were determined 24 hours post dose.

In all three pilot studies, treatment with anti-PTH1R antibody Ab328reduced tumor-induced hypercalcemia by >3 mg/dL, suggesting that theanti-PTH1R antibodies are also useful to treat cancers in which tumorsresult in elevated in vivo calcium levels in a subject.

Efficacy of Anti-PTH1R mAb, XPA.85.349, on Mouse Colon 26 Tumor-RelatedHypercalcemia

XPA.85.349 (Ab349), an expression-optimized variant of XPA.85.288, wasevaluated in the mouse colon 26 model of Tumor-related Hypercalcemia.Six week old CDF1 female mice, or Athymic Nude (NCr-nu/nu) mice wereused in the study. Colon 26 murine colon tumor cells were cultured andeach mouse were injected subcutaneously in the right flank with sevenmillion, five hundred thousand cells (7.5×10⁶) C26 cells in 0.1 mL ofserum-free media. Serum calcium was measured regularly untilhypercalcemia was observed (>12 mg/dL), usually when tumors were >1000mg. Mice were then treated with Ab349 at 2 or 6 mg/kg intravenously, anda separate group of mice with tumors >1000 mg was treated with theisotype (negative control; anti KLH G2) at 6 mg/kg intravenously. Tumorweight and serum samples were collected at pretreatment, 24, 48 and 72hours post-treatment. Total calcium levels were determined 24, 48, 72and 120 hours post treatment. Tumor weights (mg) are calculated usingthe equation for an ellipsoid sphere (l×w²)/2=mm³, where l and w referto the larger and smaller dimensions collected at each measurement andassuming unit density (1 mm³=1 mg).

Anti-PTH1R Ab349 was found to potently reduce mouse colon 26tumor-related hypercalcemia (i.e. significantly reduce total serumcalcium) for a sustained period, up to 120 hours post dose (FIG. 20A).In this tumor model, Ab349 was capable of completely reversinghypercalcemia within 24 hours. The administration of Ab349 had nosignificant effect on body weight (FIG. 20B) or tumor weight (FIG. 20C)up to 120 hours post dose.

Levels of PTHrP and PTH in serum were measured at different time pointspost dosing with Ab349 antibodies. Antibodies were administeredintraveneously at 2 and 6 mg/kg at a period of hypercalcemia t0 (n=2-6mice/group). Blood samples were collected from retro-orbital sinus at24, 48, 72 and 120 hours post administration and the concentrations ofPTHrP (Phoenix Pharmaceuticals, Inc—EIA Kit, catalog no.: EK-056-04) andmouse PTH 1-84 (QUIDEL Mouse PTH 1-84 ELISA kit, catalog no.: 60-2305)were measured. As shown in FIG. 20D, serum PTHrP is elevated intumor-bearing mice, and although treatment with Ab349 did not appear tosignificantly change PTHrP levels, Ab349 did inhibit PTHrP activity. Incontrast however, treatment with 2 or 6 mg/kg Ab349 significantlyincreased PTH levels up to 120 hours post dose (FIG. 20E).

Serum drug concentration (SDC) measurements were performed byenzyme-linked immunosorbent assay (ELISA). ELISA assays were performedas follows, ELISA plates were coated with 1 μg/mL goat anti-human Fc inPBS, plates were blocked with PBS/0.05% tween/1% BSA for 1-4 hours atroom temperature. Serum samples at various dilutions in PBS/tween/BSAadded and incubated 37° C. for 1 hr (in the final 5 minutes, 0.2 μg/mlbiotin SP-conjugated Goat anti-human IgG(Fab′) was added). Plates weresubsequently rinsed with PBS/tween, incubated with Pierce p-NitrophenylPhosphate, Disodium Salt (pNPP) in 1×DEA buffer for 1 hr at roomtemperature. The colorimetric reaction was stopped by the addition of 1NNaOH. Plates were read at 405 nM on a spectrophotometer. SDCmeasurements showed normal excretion of Ab349 over a 120 hour period.

Several pharmacokinetic measurements were performed to create apharmacokinetic profile of Ab349 following intravenous injection. CDF1female mice were administered a single dose bolus of Ab349 at 2 mg/kg or6 mg/kg. Blood samples were collected from retro-orbital sinus at 24,48, 72 and 120 hours after the administration of Ab349 and itsconcentration measured by ELISA. Data were analyzed using WinNonlin. Thepharmacokinetic parameters following 2 or 6 mg/kg Ab349 are shown in(Table 10). Ab349 was shown to have a half-life of up to 48 hrs in micewhen given at 6 mg/kg.

TABLE 10 pK Parameters For Ab349 Time (hr) 2 mg/kg 6 mg/kg Co 7.2 47.8ug/ml AUClast 414 1832 ug*hr/ml AUCinf 438 2116 ″ t½ 26 48 hr Vz 172 195ml/kg Cl 4.6 2.8 ml/hr/kg

In summary this highly potent PTH1R receptor antagonist antibody has thepotential to become a valuable therapeutic agent in a variety ofindications including hyperparathyroidism, humoral hypercalcemia ofmalignancy, and, potentially, the PTHrP-mediated cachexia seen in somecancers. Furthermore, the Ab349 antibody was effective at treatingmodels of hypercalcemia at <2 mg/kg, with a<6 mg/kg maximum dose and anextended duration of action (i.e. lasting longer than the half-life).

Numerous modifications and variations in the disclosure as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the disclosure.

1. An antibody that binds parathyroid hormone receptor 1 (PTH1R) with an affinity K_(d) of 2×10⁻⁸ M or less.
 2. The antibody of claim 1 wherein the antibody binds the N-terminal portion of PTH1R.
 3. The antibody of claim 1 wherein the antibody does not bind parathyroid hormone receptor 2 (PTH2R).
 4. The antibody of claim 1, wherein the antibody binds PTH1R on the surface of a cell.
 5. The antibody of claim 1, wherein the antibody binds allosterically to PTH1R.
 6. The antibody of any claim 1, wherein the antibody is a negative modulator antibody, optionally wherein the antibody is capable of weakening the binding affinity between PTH or PTHrP and with PTH1R by at least about 2-fold, optionally up to 1000-fold.
 7. The antibody of claim 1, wherein the antibody inhibits calcium flux in a cell in response to stimulation of the receptor with parathyroid hormone (PTH) or parathyroid hormone related protein (PTHrP).
 8. The antibody of claim 1, wherein the antibody inhibits PTH- or PTHrP-mediated cyclic adenosine mono-phosphate (cAMP) accumulation.
 9. The antibody of claim 1 that is a monoclonal antibody.
 10. An antibody that binds parathyroid hormone receptor 1 (PTH1R) comprising (a) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99,102, or a variant thereof in which one or two amino acids have been changed; (b) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 that is from the same heavy chain variable region as (a), or a variant thereof in which one or two amino acids have been changed; and (c) a heavy chain CDR3 amino acid sequence set forth SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that is from the same heavy chain variable region as (a), or a variant thereof in which one or two amino acids have been changed.
 11. An antibody that binds parathyroid hormone receptor 1 (PTH1R) comprising: (a) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variant thereof having at least 70% identity thereto; (b) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 that is from the same heavy chain variable region as (a), or a variant thereof having at least 70% identity thereto; and (c) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 that is from the same heavy chain variable region as (a), or a variant thereof having at least 70% identity thereto.
 12. An antibody that binds parathyroid hormone receptor 1 (PTH1R) comprising: (a) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, or a variant thereof having at least 70% identity thereto; (b) an independently selected heavy chain CDR2 amino acid sequence set forth in SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, or a variant thereof having at least 70% identity thereto; and (c) an independently selected heavy chain CDR3 amino acid sequence set forth in SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or a variant thereof having at least 70% identity thereto.
 13. The antibody of claim 10, wherein at least two of the heavy chain CDR1, CDR2 or CDR3 amino acid sequences are set forth in any one of SEQ ID NOs: 27-104.
 14. The antibody of claim 10, wherein three of the heavy chain CDR1, CDR2 and CDR3 amino acid sequences are set forth in any one of SEQ ID NOs: 27-104.
 15. The antibody of claim 10, that comprises an amino acid sequence at least 85% identical to a heavy chain variable region amino acid sequence set forth in SEQ ID NOs: 1-26.
 16. (canceled)
 17. The antibody of claim 10, comprising a polypeptide sequence having an amino acid sequence at least 70% identical over all three HCDRs in a heavy chain variable region, the amino acid sequences of HCDR1, HCDR2 and HCDR3 set forth in any one of SEQ ID NOs: 27-104.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. The antibody of claim 10, that comprises: (a) a light chain CDR1 amino acid sequence set forth in SEQ ID NOs: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or a variant thereof in which one or two amino acids have been changed; (b) a light chain CDR2 amino acid sequence set forth in SEQ ID NOs: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 that is from the same light chain variable region as (a), or a variant thereof in which one or two amino acids have been changed; and (c) a light chain CDR3 amino acid sequence set forth in SEQ ID NOs: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 that is from the same light chain variable region as (a), or a variant thereof in which one or two amino acids have been changed.
 23. The antibody of claim 10 that comprises: (a) a light chain CDR1 amino acid sequence set forth in SEQ ID NOs: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, or a variant thereof in which one or two amino acids have been changed; (b) an independently selected light chain CDR2 amino acid sequence set forth in SEQ ID NOs: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, or a variant thereof in which one or two amino acids have been changed; and (c) an independently selected light chain CDR3 amino acid sequence set forth in SEQ ID NOs: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, or a variant thereof in which one or two amino acids have been changed.
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. The antibody of claim 22 comprising a polypeptide sequence having an amino acid sequence at least 70% identical over all three LCDRs of a light chain variable region, the amino acid sequences of LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 131-208.
 30. The antibody of claim 22 comprising (i) an amino acid sequence at least 70% identical over all three LCDRs, of a light chain variable region, the amino acid sequences of LCDR1, LCDR2 and LCDR3 set forth in any one of SEQ ID NOs: 131-208 and (ii) an amino acid sequence at least 70% identical over all three HCDRs of a heavy chain variable region, the amino acid sequences of HCDR1, HCDR2 and HCDR3 set forth in any one of SEQ ID NOs: 27-104.
 31. An antibody that binds parathyroid hormone receptor 1 (PTH1R) comprising a light chain variable region and/or a heavy chain variable region, wherein (a) the light chain variable region comprises at least a CDR1 selected from SEQ ID NOs: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 or sequences at least 80% identical thereto, a CDR2 selected from SEQ ID NOs: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or sequences at least 80% identical thereto, and/or a CDR3 selected from SEQ ID NOs: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 or sequences at least 80% identical thereto; and/or wherein (b) the heavy chain variable region comprises at least a CDR1 selected from SEQ ID NOs: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102 or sequences at least 80% identical thereto, a CDR2 selected from SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 or sequences at least 80% identical thereto, and/or a CDR3 selected from SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 80% identical thereto.
 32. The antibody of claim 31, wherein (a) the light chain variable region comprises at least a CDR1 selected from SEQ ID NO: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 or sequences at least 90% identical thereto, a CDR2 selected from SEQ ID NO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or sequences at least 90% identical thereto, and a CDR3 selected from SEQ ID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 or sequences at least 90% identical thereto; and/or wherein (b) the heavy chain variable region comprises at least a CDR1 selected from SEQ ID NO: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102 or sequences at least 90% identical thereto, a CDR2 selected from SEQ ID NO: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 or sequences at least 90% identical thereto, and a CDR3 selected from SEQ ID NO: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 90% identical thereto.
 33. The antibody of claim 31, wherein (a) the light chain variable region comprises at least a CDR1 selected from SEQ ID NO: 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206 or sequences at least 90% identical thereto, a CDR2 selected from SEQ ID NO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or sequences at least 90% identical thereto, and a CDR3 selected from SEQ ID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208 or sequences at least 90% identical thereto; and/or wherein (b) the heavy chain variable region comprises at least a CDR1 selected from SEQ ID NO: 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102 or sequences at least 90% identical thereto, a CDR2 selected from SEQ ID NO: 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 or sequences at least 90% identical thereto, and a CDR3 selected from SEQ ID NO: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104 or sequences at least 90% identical thereto.
 34. (canceled)
 35. The antibody of claim 22, in which one or more light chain framework amino acids have been replaced with corresponding amino acid(s) from another human antibody amino acid sequence, optionally wherein the framework comprises one or more of the changes set out in FIG.
 21. 36. The antibody of claim 22, wherein the antibody is selected from the group consisting of XPA.85.012, XPA.85.017, XPA.85.288, XPA.85.388, XPA.85.389 and XPA.85.390.
 37. (canceled)
 38. (canceled)
 39. An antibody of claim 10 that binds parathyroid hormone receptor 1 (PTH1R) with an affinity K_(d) of 10⁻⁶ M or less.
 40. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain or light chain of claim
 1. 41. An expression vector comprising the nucleic acid molecule of claim 40 operably linked to an expression control sequence.
 42. A host cell comprising the vector of claim
 41. 43. The host cell of claim 42, comprising a nucleic acid molecule encoding a heavy chain and a light chain variable region, wherein the heavy chain and light chain nucleic acids are expressed by different nucleic acids or on the same nucleic acid.
 44. A method of using the host cell of claim 42 to produce an antibody, comprising culturing the host cell of claim 42 under suitable conditions and recovering said antibody.
 45. (canceled)
 46. A sterile pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.
 47. A method for treating hypercalcemia associated with increased parathyroid hormone or parathyroid hormone related protein expression comprising the step of administering to a subject in need thereof a therapeutically effective amount of an antibody of claim 1 or a pharmaceutical composition of claim
 46. 48. A method for treating a disease, condition or disorder associated with increased parathyroid hormone expression, increased parathyroid hormone related protein expression or parathyroid hormone receptor 1 (PTH1R) expression comprising the step of administering to a subject in need thereof a therapeutically effective amount of an antibody of claim 1 or a pharmaceutical composition of claim
 46. 49. The method of claim 48, wherein the disease, condition or disorder is selected from the group consisting of cancer, PTH- or PTHrP-induced hypercalcemia, Humoral Hypercalcemia of Malignancy (HHM), familial hypocalciuric hypercalcemia, tuberculosis, sarcoidosis, Primary Hyperparathyroidism (PHPT), Secondary Hyperparathyroidism (SHPT) and cachexia.
 50. The method of claim 49 wherein the disease is PHPT and the subject is a non-surgical patient or a patient who has failed surgery.
 51. The method of claim 49 wherein the disease is SHPT and the subject has chronic kidney disease.
 52. The method of claim 47 wherein the administration reduces the incidence of cancer metastasis in the subject compared to a subject not receiving the antibody.
 53. The method of claim 52 wherein the metastasis includes metastasis to the bone or skeletal tissues, liver, lung, kidney or pancreas.
 54. The method of claim 47 wherein the administration ameliorates one or more symptoms of hypercalcemia.
 55. The method of claim 47, wherein the administration extends HHM survival due to reduced hypercalcemia and/or wasting syndrome.
 56. The method of claim 47 wherein the antibody is administered intravenously, intraarterially, intraperitoneally, intramuscularly, intradermally or subcutaneously.
 57. The method of claim 47 wherein the antibody is administered in combination with a second agent.
 58. The method of claim 47 wherein the antibody is administered once per week, once every 2 weeks, twice per month, once monthly, once every two months, or once every three months.
 59. A composition comprising an antibody of claim 1 or a pharmaceutical composition of claim 46 for use in treating a condition or disorder associated with increased parathyroid hormone expression or increased parathyroid hormone related protein expression.
 60. A composition comprising an antibody of claim 1 or a pharmaceutical composition of claim 46 for use in treating a condition or disorder associated with hypercalcemia. 